© 2001 American Heart Association, Inc.
Molecular Medicine |
Chimeric Smooth Muscle–Specific Enhancer/Promoters
Valuable Tools for Adenovirus-Mediated Cardiovascular Gene Therapy
From the Cardiovascular Gene Therapy Laboratory (S.R., P.N., F.A., V.C.), Transgène S.A., Strasbourg, France; Department of Pathology (A.M.-N.), Hôpital de Hautepierre, Strasbourg, France; Molecular Biology of Differentiation Laboratory (A.P., D.P.), D. Diderot University, Paris, France; and Department of Pathology (G.G.), University of Geneva-CMU, Geneva, Switzerland.
Correspondence to Dr Valerie Calenda, Transgène S.A., Cardiovascular Gene Therapy Laboratory, 11 rue de Molsheim, 67082 Strasbourg, Cedex, France. E-mail calenda{at}transgene.fr
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Abstract |
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Abstract—Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22
promoter compared
with the viral cytomegalovirus (CMV) enhancer/promoter. We have
generated chimeric transcriptional cassettes containing either a SM
(SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer
combined with the SM22 promoter. With both constructs we observed
significantly stronger expression that remains SM-specific. In vivo,
reporter gene expression was restricted to arterial SMCs with no
detectable signal at remote sites. Moreover, when interferon-
expression was driven by one of these two chimeras, SMC growth was
inhibited as efficiently as with the CMV promoter. Finally, we
demonstrate that neointima formation in the rat carotid balloon injury
model was reduced to the same extent by adenoviral gene transfer of
interferon- driven either by the SM-myosin heavy chain
enhancer/SM22 promoter or the CMV promoter. These results indicate
that such vectors can be useful for the treatment of hyperproliferative
vascular
disorders.
Key Words: smooth muscle myosin • creatine kinase • interferon- • restenosis • gene transfer
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Smooth muscle cell (SMC) migration and proliferation are key factors in the development of atherosclerosis and restenosis after angioplasty or stent placement.1 These processes are associated with phenotypic modulation of SMCs characterized by modification of gene expression.2 Hence, the overexpression of an individual gene in vascular SMCs by local gene transfer is considered a promising therapeutic approach. Several vector systems have been developed to facilitate gene delivery into SMCs. Among them, the most widely used are recombinant adenovirus vectors (Ads). In SMCs, Ads have been used successfully to impair cell growth in vitro and to diminish intimal hyperplasia in animal models of restenosis.3 However, because systemic administration of a high viral dose is associated with cytotoxic effects in nontarget tissues, the clinical application of Ads can be compromised.4 To solve these problems, several strategies have been considered. A local delivery and the retargeting of the vectors may reduce viral dissemination. Moreover, modification of the expression cassette by addition of cell type–specific transcriptional elements may ensure targeted protein expression and reduce host immune responses to transgenic proteins.5 The replacement of viral promoters by SM-specific cis-acting sequences has been studied previously.6 7
The transcriptional regulation of SM-specific genes has been
centered on structural proteins (actin and myosin) and actin-binding
proteins (SM22 and
calponin).8 The SM-myosin
heavy chain (MHC), a major contractile protein and a powerful marker
for the study of SMC differentiation, is regulated by multiple
cis-acting elements, including
CarG boxes, that have been shown to be key regulators for SM gene
transcription.9 SM22, a
calponin-related protein that is expressed specifically in SM,
possesses 3 CarG boxes in its promoter that are sufficient to direct
arterial tissue–specific
expression.6 10
The limiting factor for the use of tissue-specific promoters is the low
level of expression compared with their viral counterparts. To our
knowledge, no data related to the level of SM22-driven gene
expression are available in an adenoviral context. Hence, in the
present study, we evaluated the strength of the SM22 promoter and
revealed a low efficiency by comparison with the cytomegalovirus (CMV)
promoter. Two strategies have been described to obtain stronger
artificial tissue-specific promoters. One consists of the construction
of synthetic promoter libraries by random combination of
tissue-specific regulatory
elements.11 Another is to
exploit the endogenous genomic sequences that enhance tissue-specific
expression. We have explored this latter strategy by fusing the SM22
promoter either with the SM-MHC enhancer or with the creatine kinase
enhancer.12 13 In
vitro, we demonstrated that these new transcriptional cassettes were
more effective than the SM22 promoter alone. In addition, they
improved the cell type–specific expression of the reporter gene. In
vivo, when injected intravenously in mice, no expression was observed
in nontarget tissues. Finally, when tested in the rat carotid balloon
injury model, the expression of interferon- (IFN-) leads to a
biological effect when driven either by one of the enhancer/promoter
combinations (SM-MHC/SM22) or by the CMV promoter. Taken together,
our data indicate that these chimeric transcriptional elements are
excellent candidates for a targeted vascular gene
therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells and Culture Conditions
Rat SMCs were isolated from aortas (ratAo) and from injured aortas 15 days after balloon catheter deendothelialization (ratIT15).14 The human pulmonary epithelial A549 and the murine myoblast C2C12 cell lines were purchased from the American Type Culture Collection (CCL-185 and CRL-1772; Manassas, Va). The rat intestinal epithelial cells (IEC18) were kindly provided by Dr Simon-Assmann (INSERM U381, Strasbourg, France). Cells were cultured in DMEM containing 10% FCS (Life Technologies) supplemented with 0.2 U/mL insulin (Sigma-Aldrich) for IEC18. During and after adenoviral infection, cells were maintained in 2% FCS–containing medium, except C2C12, which were maintained in 10% FCS to prevent fusion.
Adenovirus Construction, Production, and
Titration
Expression cassettes were obtained by insertion in
the adenoviral E1-deleted region of the different promoters and
enhancers followed by an intron and a transgene, as listed in the
Table
.
All viral vectors were constructed as infectious plasmids by homologous
recombination in Escherichia
coli BJ5183.15
They were all deleted for the E1 and E3 regions. Adenoviral plasmids
were digested by PacI and
transfected in the 293 complementation cell line. After virus
propagation and purification, infectious units (iu) were
titrated.
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In Vitro Experiments
The susceptibility to adenoviral infection was
determined for each cell type using various multiplicities of infection
(MOIs) of AdCMVeGFP. Cells were infected at day 1 (D1) and harvested at
D2, and the percentage of expressing cells was determined by
quantitative analysis of enhanced green fluorescent protein
(eGFP) expression by flow cytometry (FACSCalibur, Becton Dickinson
Biosciences). In other experiments, cells were infected and harvested
at D4 to allow eGFP accumulation. For the duration of expression
experiment, cells were seeded at a density of
1.105 cells/well in 6 well plates (Falcon,
Becton Dickinson) at D0 and infected with either AdCMVeGFP or
AdSM-MHC/SM22eGFP at D1. They were maintained up to D15 in 2%
FCS–containing medium without medium replacement and harvested at
different time points to quantify eGFP expression. The strength of the
different regulatory sequences was measured using the global
fluorescence index (GFI) calculated as the product of the percentage of
eGFP-positive cells by the mean fluorescence
value.16 For the
cell-differentiation experiment, infections were performed as described
above and then cells were washed and placed in either 2%
FCS–containing or 10% FCS–containing medium and harvested at D4. For
cell growth inhibition experiment, cells were seeded at a density of
3.104 cells/well in 6 well plates at D0 and
infected with AdSM-MHC/SM22IFN and AdCMVIFN at D1. Cells were
counted at D5. A sample of the culture medium was harvested at D4 to
quantify the secreted rat IFN- (Quantikine M rat IFN-, R&D
Systems).
In Vivo Gene Transfer Into Mice
All animal experiments were performed in a special
pathogen-free facility and were conducted according to the French
regulations for animal experimentation (Decret No. 87-848, 19.10.1987).
Nine-week-old female immunocompetent mice were used for experiments
(C57BL/6, Iffa-Credo, L’Arbresle, France). At D0, adenoviruses
were injected intravenously at 2.109 iu.
Mice were killed at D3, and organs (liver, lungs, spleen, and heart)
were harvested and fixed in PBS containing 2% formaldehyde. The eGFP
expression was evaluated by fluorescence
microscopy.
In Vivo Gene Transfer Into Rat Carotid
Adult male Wistar rats (body weight 400g) were used
for experiments (Iffa-Credo). The left common carotid artery was
injured by balloon
catheterization.17 Then a
1-cm-long segment of the injured carotid was isolated and flushed with
0.2 mL NaCl 0.9%. Adenovirus (50 µL)
(2.109 iu) was allowed to dwell for 5
minutes. Rats were killed at different time points according to the
experiments. Vessels were fixed with either 2% or 4% formaldehyde in
PBS at normal blood pressure. Then carotids were excised and treated
for the different histological analyses.
Histological and Immunocytochemical
Analyses
SM22 immunohistochemical staining was performed on
4-µm-thick sections. Deparaffinized sections were incubated with the
E-11 SM22 mouse monoclonal antibody (kindly provided by Drs A.
Chiavegato and S. Sartore, University of Padua, Padua, Italy) at
a dilution of 1:100.18 The
presence of SM22 was revealed by means of the
streptavidin-biotin-complex peroxidase method (LSAB kit, Dako). For
ß-galactosidase activity, vessels fixed in 2% formaldehyde were
incubated for 24 hours at 37°C in 5 mmol/L
K3Fe (CN6), 5 mmol/L
K4Fe (CN6), 2 mmol/L
MgCl2, and 1 mg/mL
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.
Five-micrometer sections were examined for blue nuclear staining. For
morphological analyses, 5-µm sections from 4% formaldehyde–fixed
arteries were stained with H&E, and the medial and intimal areas were
evaluated by image analysis using an Olympus DP11 camera and AnalySIS
software (Soft Imaging System GmbH).
Statistical Analysis
All results are expressed as mean±SEM and were
analyzed by Student’s t test.
Differences were considered statistically significant at values of
P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Vitro Evaluation of SM22
Promoter
Strength and SpecificityThe susceptibility to adenovirus infection of ratAo, ratIT15, IEC18, and A549 cells was determined by infection with AdCMVeGFP at MOIs ranging from 1 to 1000. The MOI corresponding to 100% of eGFP-expressing cells was determined by flow cytometry. A549 cells, IEC18, and ratIT15 but not ratAo were efficiently infected at relatively low MOIs (A549: MOI 50; IEC18: MOI 50; ratIT15: MOI 10; ratAo: MOI 300). These data revealed that SMCs have a different susceptibility to adenoviral infection according to their phenotype.
To better compare eGFP expression driven either by the
SM22 promoter or the CMV promoter, the level of eGFP was analyzed by
flow cytometry 3 days after infection. We observed that only a fraction
of AdSM22eGFP-infected SMCs expressed the reporter gene. The
fluorescence intensity of this fraction was determined, but we decided
to express the results as the mean of fluorescence of the global
population. This better reflects the total amount of transgene that can
be produced by one expression cassette in a given cell type. Thus, the
promoter strength was evaluated using the
GFI.16 We observed that the
GFI of AdSM22eGFP-infected cells was stronger in ratAo than in ratIT15.
A weak background was present in the 2 non–SM cell lines from rat
intestine (IEC18) and human lung (A549)
(Figure 1A). Interestingly, when compared with AdCMVeGFP (GFI
arbitrarily set at 100%), the AdSM22eGFP-related expression was nearly
identical in ratIT15 and ratAo (8±1.8% versus 5.7±0.7%,
Figure 1B), reflecting a similar regulation of both
promoters in a defined SMC population. However, the SM22
promoter–related expression remained relatively
weak.
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Influence of the MOI on SM22
Promoter– Driven Expression
The comparison of the CMV- and the SM22-driven
expression levels in SMC populations was done with different viral
doses according to their susceptibility to adenoviral infection. We
cannot rule out that the viral load influences the cell physiology and
consequently the strength of the promoters. Such interactions were
demonstrated in primary human
SMCs.19 To evaluate the
effect of high viral loads on transgene expression, ratAo and ratIT15
were infected at an MOI higher than the viral dose that allowed 100%
of infection. In ratAo, increasing the viral dose from MOI 300 to MOI
500 resulted in a 5-fold increase of the SM22-driven expression,
whereas the CMV-driven expression remained unchanged
(Figure 2). In ratIT15, the CMV- and SM22-driven eGFP
expression increased with the MOI. Thus, the comparison of viral and
SM-specific promoters should be done without superinfection, because
they are not equally affected by the viral dose.
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Expression of the SM22 Protein During
Neointima Formation in Rat Carotid Arteries
Like natural transcription units, the regulation of
tissue-specific expression cassettes in the adenoviral context depends
largely on the presence of the required transcription factors. We
hypothesized that the regulation of the SM22-driven expression
cassette will depend on the regulation of the endogenous SM22,
because, like the SM-MHC or the SM -actin, SM22 is a SMC
transcriptionally regulated differentiation
marker.20 Therefore, we
determined the pattern of expression of SM22 during intimal
thickening formation in the rat model of carotid injury. In uninjured
rat carotid, SM22 was present throughout the media
(Figure 3A). After injury, we did not observe a
downregulation of SM22 in the media at any time point analyzed
(Figures 3B through 3E). SM22 was expressed during
neointima formation in migrating and proliferating SMCs
(Figures 3C through 3E). We therefore confirmed that the
LacZ bacterial gene driven by
the SM22 promoter could be expressed in this model of arterial
injury. As previously
reported,6 we observed a blue
nuclear staining in cells scattered throughout the media
(Figure 3F).
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In Vitro Evaluation of the Strength and
Specificity of Chimeric Regulatory Sequences
In vitro experiments revealed the weakness of the
SM22 promoter and suggested that it could not drive production of
sufficient amounts of therapeutic molecules in a clinical setting. This
assertion is reinforced by the rather low efficiency of infection
reported with classical catheter-based gene delivery techniques.
Improvements of promoter strength may be achieved by the addition of
enhancers from viral, cellular, or synthetic
origin.11 21 We
decided to use enhancers belonging to the second group.
The creatine kinase enhancer (CKenh) was chosen for its
known muscle specificity and the SM-MHC enhancer (SM-MHCenh) for its SM
specificity. The strength and specificity of expression of these
chimeric regulatory elements were tested in ratAo, ratIT15, and A549
cells. The chimeric constructs were more specific than the SM22
promoter alone, because eGFP expression was 4-fold weaker in A549 cells
with both the CKenh/SM22 and SM-MHCenh/SM22 promoters
(Figure 4A). In addition, all SM22-based promoters gave
similar results in IEC18 (data not shown). The CKenh increased the
level of expression by about 3-fold in ratAo and ratIT15, with
13.2±5% and 21.3±3% of the CMV-related expression, respectively.
The SM-MHCenh showed the same pattern of expression except that it was
slightly stronger (not statistically significant) in both SMC
populations, with 16.9±4% and 23.5±2.8% of the CMV-related
expression.
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Because of the skeletal muscle origin of the CKenh, we
tested the corresponding chimeric vector in murine myoblasts using the
C2C12 cell line. Surprisingly, we did not observe a strong expression
with the CKenh/SM22 promoter. The background attributable to the
SM22 promoter (1.7% of the CMV promoter) was increased by the CKenh
(5.6%), but the expression level remained 3 to 4 times lower than in
SMC populations (data not shown).
Thus, in vitro experiments indicate that the CKenh/SM22
and the SM-MHCenh/SM22 expression cassettes increase transgene
expression and maintain specificity.
Effect of Cell Differentiation on Chimeric
Promoter-Related Expression
Because it was reported that serum concentration has an
impact on the SM22 promoter–driven
LacZ expression in plasmid
transient transfections,10
we evaluated the effect, with Ad, of serum-induced SMC
dedifferentiation on SM22 promoter–driven, CKenh/SM22
promoter–driven, and SM-MHCenh/SM22 promoter–driven expression.
AdCMVeGFP was used as positive control. Only ratAo cultured in 2% or
10% FCS were infected, because it is well accepted that ratIT15 are
undifferentiated in reduced-serum
conditions.22 We observed
that the SM22 promoter–driven, CKenh/SM22 promoter–driven, and
SM-MHCenh/SM22 promoter–driven eGFP expressions were decreased in
high serum concentration
(Figure 4B
). This result might suggest that the chimeric
promoter containing two SM-specific regulatory sequences (SM-MHC and
SM22) follows, such as the SM22 promoter alone, the normal
regulation of the corresponding differentiation markers, known to be
more highly expressed in differentiated cells. However, the
muscle-specific CKenh did not modify the high-serum-induced
downregulation of eGFP expression when associated with the SM-specific
SM22 promoter. More surprisingly, the CMV-driven expression, when
tested in both culture conditions, was also regulated by serum
concentration. Taken together, these data indicate that the strength of
viral, muscle, and SM-specific regulatory sequences is similarly
downregulated after SMC dedifferentiation.
In Vivo Evaluation of Chimeric Vectors in
Mice
Dissemination of adenoviral vectors after intravenous
or intra-arterial administration is a major problem for their use as
therapeutic gene vehicles. Therefore, we verified that SM22
promoter–driven expression did not occur in any of the main adenovirus
natural target organs (ie, liver, lungs, spleen, and heart). This
experiment was done by adenovirus injection into C57BL/6 mice for
technical facilities. AdCMVeGFP and Tris-HCl injections were used as
positive and negative controls. Four mice were injected intravenously
with 2.109 iu of each construct, and the
presence of eGFP expression in the different organs was determined by
fluorescence microscopy. The Southern blot performed on the DNA
extracted from the 4 main organs indicated that all mice were correctly
infected (data not shown). The 4 mice injected with AdCMVeGFP showed a
significant fluorescence in all organs tested, whereas mice injected
with SM22-containing vectors or the two chimeric promoter-containing
vectors did not show any fluorescence, confirming, therefore, the
tissue specificity of these constructs (data not
shown).
Duration of the Expression Using
AdSM-MHC/SM22eGFP
Even if similar results were obtained with both
chimeric constructs in terms of strength, specificity, and regulation,
we decided to concentrate on the SM-specific cassette. Before studying
the efficacy of a combination between this sequence and a therapeutic
gene, we addressed the question of the duration of expression driven by
the SM-MHCenh/SM22 promoter in vitro in ratAo. Indeed, the high
proliferation rate of ratIT15 does not allow their use in long-term
experiments, because they rapidly become confluent even in low-serum
conditions.22 After a
maximum reached at D4, the expression decreased with both the
SM-MHCenh/SM22 and the CMV promoters, albeit at different rates
(Figure 5). However, 35% of the cell population still
expressed the eGFP at D15 when driven by the SM-MHCenh/SM22
promoter, confirming, therefore, the interest of this sequence to drive
a SM-specific expression cassette.
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Effect of AdSM-MHC/SM22IFN on SMC
Proliferation In Vitro
We analyzed the capacity of the rat IFN- gene driven
by the SM-MHCenh/SM22 promoter to prevent SMC proliferation. We used
the ratIT15, which better represents the target cells in the treatment
of restenosis. AdSM-MHC/SM22IFN was compared with AdCMVIFN and
with AdCMVeGFP used as negative control. Results are expressed as the
percentage of growth inhibition 4 days after infection.
The chimeric promoter generated a 54±5.2% growth
inhibition versus 59±7.3% for the CMV
(Figure 6A). The difference is not statistically significant
(P=0.37). The SMC growth
inhibition attributable to AdCMVeGFP was small (9±12.1%) and not
statistically significant
(P=0.41) compared with
noninfected cells.
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Supernatants of infected cells were harvested at D4 and
analyzed by ELISA to determine IFN- concentration. The
SM-MHCenh/SM22 promoter gave rise to an IFN- concentration of
8.7±13.5 ng/mL versus 31.2±20.8 ng/mL
(P=0.026) for the CMV promoter
(Figure 6B). No detectable levels of IFN- were obtained in
noninfected and AdCMVeGFP-infected cells.
Effect of AdSM-MHC/SM22IFN on Neointima
Formation in the Rat Carotid Model
We finally examined the effect of AdSM-MHC/SM22IFN
in the rat model of carotid injury. Rats were locally injected with
2.109 iu of AdSM-MHC/SM22IFN,
AdCMVIFN, Adnull, AdCMVeGFP, and AdSM-MHC/SM22eGFP subsequently to
balloon catheter injury, and arteries were analyzed after 14 days. In
Adnull-, AdCMVeGFP-, and AdSM-MHC/SM22eGFP-treated control rats,
extensive intimal thickening was observed in all injured vessels with
no statistically significant differences (0.106±0.023
mm2, 0.090±0.021
mm2, and 0.085±0.018
mm2, respectively
Figures 7A and 7D). In contrast, AdCMVIFN- and
AdSM-MHC/SM22IFN-treated rats showed a similar 25% to 35%
reduction in the cross-sectional area of the carotid neointima
(0.070±0.051 mm2
[Figure 7B] and 0.057±0.009 mm2
[Figure 7C], respectively). The media was not affected by
either adenoviral vector treatments (0.113±0.011
mm2 and 0.109±0.013
mm2 versus 0.119±0.015
mm2, 0.114±0.013
mm2, and 0.115±0.014
mm2,
respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously demonstrated that adenoviral vectors could be transcriptionally targeted to vascular SMCs.6 However, the use of tissue-specific promoters is a matter of debate, because they are generally considered to be weak. Thus, there is still a need to develop efficient, strong, and specific SM regulatory sequences allowing the production of therapeutic proteins in sufficient amounts. In the present study, we show that a chimeric construct containing a SM-specific enhancer associated with the SM22
promoter meets these
criteria. We first confirmed that the SM22 promoter alone is
relatively weak, reaching only 8% of the CMV-promoter strength.
Previous studies indicate that the SM22 promoter in a plasmid
context was as strong as the RSV
promoter.10 This is
consistent with our unpublished observations, showing that the Rous
sarcoma virus (RSV) promoter is about 10% as strong as the CMV
promoter. However, it is important to note that results obtained with
adenoviral vectors may vary according to SMC origin. We observed that
ratAo are less sensitive to adenoviral infection but more capable of
expressing transgenes under the control of the SM22 promoter than
ratIT15. When these SMC populations are exposed to a SM22-containing
adenovirus, only a fraction of infected cells expresses the reporter
gene, suggesting that SMC populations are heterogeneous as far as their
capacity of transactivating this SM-specific promoter is concerned. We
also observed a heterogeneous response to infection using the viral CMV
promoter, because increasing the viral load generated stronger
expression only in ratIT15. It has been shown elsewhere that nuclear
factor-
B, known to be an important transcription factor required for
CMV promoter activity, is present in SMCs involved in response to
balloon
injury.23
Because the SM22 is a transcriptionally regulated
differentiation marker, we hypothesized that the expression cassette
and the endogenous promoter would be simultaneously activated in
vivo.20 In contrast to the
SM22 downregulation reported in the rabbit model, the endogenous
protein was present at all time points in both medial and neointimal
SMCs of the injured rat
carotid.18 These data
confirm the interest of an SM22 promoter–driven expression cassette
for sustained in vivo expression in synthetic and contractile
SMCs.
Despite interesting properties, such as in vitro and in vivo
cell lineage–restricted expression, the SM22 promoter remains weak.
Therefore, we improved the expression cassette using the SM-MHC and the
CK enhancers. They were both efficient despite their heterologous
origin, rabbit for SM-MHCenh and human for CKenh, and increased the
SM22 promoter–related expression about 3-fold. These enhancing
effects were in the same range as those observed with other
promoters.12 24
In addition, we observed that these enhancers maintained the
specificity of expression and did not change the SM22 promoter
regulation, because the stronger transcriptional activity was obtained
with the more differentiated cells.
To our knowledge, this is the first time that the CKenh has
been tested in SMCs alone or in combination with a SM-specific
promoter. It is surprising that the CKenh, usually defined as a
striated muscle-specific enhancer, had a SM-specific effect. We could
hypothesize that adenoviral sequences have an impact on the CKenh and
that they redirect its specificity. Such interactions between viral
sequences and a specific enhancer/promoter chimera containing the CKenh
were previously described.24
On the other hand, the CKenh contains functional domains involved in
the regulation of genes from skeletal, cardiac, and SM origin. These
muscle-specific motifs could be responsible for the activation of the
CKenh in an SM context. Indeed, the SM22 promoter CarG boxes lie
closer to the CKenh CarG motif, because we placed the enhancer in an
antisense orientation shown to be responsible for a stronger
expression.13 It has already
been shown that SM-selective expression depends on the positioning of
CarG elements and cooperative interactions between
them.25 Confirming this
hypothesis, no expression was observed in skeletal muscle after
intramuscular injection in mice of the CKenh-containing construct.
Taken together, these results indicate that both chimeric constructs
are potentially useful for vascular gene transfer strategies. However,
we chose the SM-MHCenh/SM22 promoter for its strict SM-specific
sequence composition.
The duration of gene expression, studied with this
regulatory sequence, revealed a rapid decrease of the eGFP expression
compared with the CMV promoter–driven expression. This decrease could
be explained at least in part by the low serum condition used to
prevent SMC proliferation. It was recently demonstrated that the
SM22 promoter and SM-MHC promoter presented a 8-fold loss of their
activity in long-term serum-deprived SMCs compared with serum-fed
cells. On the contrary, the activity of a viral promoter was unaffected
by serum culture
conditions.26 Interestingly
we observed that 35% of the AdSM-MHC/SM22eGFP-infected cells still
expressed the eGFP 14 days after infection. In addition, the absence of
downregulation of the endogenous SM22 during the intimal thickening
process additionally supports the idea that conditions encountered in
vivo will favor the persistence of the expression driven by the
SM-MHCenh/SM22 promoter.
The therapeutic interest of the chimeric promoter was then
tested in combination with the rat IFN- known for its
antiproliferative
properties.27 We observed a
significant inhibition of neointimal SMC growth in vitro and a reduced
intimal thickening formation in the rat carotid model. This
adenoviral-mediated local expression avoids the administration of huge
amounts of recombinant protein, which is usually associated with side
effects. For instance, a 50% reduction of the neointima requires a 100
µg/kg IFN- treatment during 7
days.27
The CMV-driven IFN- effect was surprisingly not
significant compared with Adnull, AdCMVeGFP or AdSM-MHC/SM22eGFP
(P=0.18,
P=0.43, and
P=0.59, respectively). Several
factors, such as toxicity and inflammation, could be responsible for
the substantial variations observed in this group. We may hypothesize
that the CMV promoter produces toxic IFN- concentrations, leading to
cell death and to an inflammatory response that could increase the
number of infiltrating cells in the neointima and thus partly
counteract the beneficial effect of IFN-. By comparison, the lower
IFN- concentration produced by the SM-MHCenh/SM22 promoter could
favor the persistence of expressing cells, resulting in a more
reproducible inhibition of the neointima.
In conclusion, the present study demonstrates that
chimeric-specific regulatory elements based on the SM22 promoter are
efficient for the treatment of cardiovascular hyperproliferative
disorders. The development of new generations of adenoviral vectors
containing these chimeras could be one of the promising strategies for
a SMC targeted gene therapy devoid of side
effects.
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Acknowledgments |
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This work was supported in part by the Convention Industrielle pour la Formation par la Recherche CIFRE (grant No. 66/98). We are grateful to Drs A. Chiavegato and S. Sartore for providing the SM22
antibody. We thank Dr M. Courtney for critical
reading of the manuscript, the people of histology and animal facility
departments for the management of animal experiments, and B. Heller for
photographic work. |
Footnotes |
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Original received September 6, 2000; revision received December 29, 2000; accepted February 1, 2001.
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