© 2000 American Heart Association, Inc.
Vascular Biology |
Second-Generation Adenoviral Vectors Do Not Prevent Rapid Loss of Transgene Expression and Vector DNA From the Arterial Wall
From the Gladstone Institute of Cardiovascular Disease (S.W., D.B.S., R.M.D., G.V., A.B.S., D.A.D.), San Francisco, Calif, and the Department of Medicine and Cardiovascular Research Institute (S.W., D.A.D.), University of California, San Francisco.
Correspondence to Dr David A. Dichek, Gladstone Institute of Cardiovascular Disease/UCSF, PO Box 419100, San Francisco, CA 94141-9100. E-mail ddichek{at}gladstone.ucsf.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—The utility of adenoviral vectors for arterial gene transfer is limited by the brevity of their expression and by inflammatory host responses. As a step toward circumventing these difficulties, we used a rabbit model of in vivo arterial gene transfer to test 3 second-generation vectors: a vector containing a temperature-sensitive mutation in the E2A region, a vector deleted of E2A, and a vector that expresses the immunomodulatory 19-kDa glycoprotein (gp19k) from adenovirus 2. Compared with similar first-generation vectors, the second-generation vectors did not significantly prolong ß-galactosidase transgene expression or decrease inflammation in the artery wall. Although cyclophosphamide ablated the immune and inflammatory responses to adenovirus infusion, it only marginally prolonged transgene expression (94% drop in expression between 3 and 14 days). In experiments performed with "null" adenoviral vectors (no transgene), loss of vector DNA from the arterial wall was also rapid (>99% decrease between 1 hour and 14 days), unrelated to dose, and only marginally blunted by cyclophosphamide. Thus, the early loss of transgene expression after adenoviral arterial gene transfer is due primarily to loss of vector DNA, is not correlated with the presence of local vascular inflammation, and cannot be prevented by use of E2A-defective viruses, expression of gp19k, or cyclophosphamide-mediated immunosuppression. Adenovirus-induced vascular inflammation can be prevented by cyclophosphamide treatment or by lowering the dose of infused virus. However, stabilization of adenovirus-mediated transgene expression in the arterial wall is a more elusive goal and will require novel approaches that prevent the early loss of vector DNA.
Key Words: rabbits • gene therapy • carotid arteries • ß-galactosidase • inflammation
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The utility of adenoviral vectors for arterial gene transfer is limited by the brevity of transgene expression and by local inflammation.1 2 3 Both difficulties are believed to result from antigen-specific immune responses to adenoviral and transgene proteins.4 5 Engineering the vector genome to reduce or eliminate adenoviral protein expression (ie, production of second- and third-generation adenoviral vectors) is a commonly used strategy to circumvent destructive immune responses to adenoviral vectors. This strategy has been applied successfully in liver, lung, and skeletal muscle models.6 7 8 9 Enthusiasm for second-generation adenoviral vectors continues to be expressed in relation to cardiovascular gene transfer10 ; however, use of these vectors for arterial gene delivery has not yet been definitively reported. We recently reported the unanticipated result that adenovirus-mediated transgene expression declines rapidly in arteries of mice that lack antigen-specific immunity.11 This result suggested that paradigms developed to explain the loss of adenovirus-mediated transgene expression in other organs may not explain loss of transgene expression from the arterial wall.
To begin to test whether engineering of the adenoviral vector genome could improve arterial gene transfer, we constructed 2 second-generation adenoviral vectors. Results of experiments performed with these vectors, along with the above-cited data from immunodeficient mice,11 caused us to question whether antigen-specific immunity plays a major role in the early loss of adenovirus-mediated transgene expression after arterial gene transfer. We performed additional experiments with a third second-generation vector, "null" viruses (lacking a transgene), and cyclophosphamide (CTX) immunosuppression. These experiments suggested that adenovirus-mediated transgene expression in the arterial wall declines primarily due to early loss of vector DNA and that the antigen-specific immune system plays a relatively minor role in expediting this loss. Principles developed from adenoviral gene transfer studies performed in other organs do not appear adequate to explain the behavior of adenoviral vectors infused to the arterial wall.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
For all subheadings, additional information is available in the expanded Methods section available online at http://www.atvb.ahajournals.org
Recombinant Viruses
Of the 6 adenoviral vectors used in these experiments, 4 were
generated in our laboratory: AdRSVnLacZ,12 Adgp19kLacZ,
AdE2AtsLacZ, and AdCMVNull (Figure 1
). Adgp19kLacZ expresses the 19-kDa
glycoprotein (gp19k) from human adenovirus type
2.13 AdE2AtsLacZ contains a
temperature-sensitive mutation in the E2A DNA-binding
protein.14 AdCMVNull does not contain a transgene. Two
additional adenoviral vectors were provided by IntroGene (Leiden, the
Netherlands): AdE2A+ and AdE2A-. Both IntroGene vectors were
E1-deleted adenovirus 5-based vectors with a cytomegalovirus (CMV)
promoter (but no transgene) inserted at the site of the E1 deletion.
AdE2A- was also deleted of the E2A region.
|
Phenotypic Characterization of Second-Generation Vectors
Expression of gp19k protein by Adgp19kLacZ was detected by
indirect immunofluorescence performed after
infection of Chinese hamster ovary cells and primary rabbit smooth
muscle cells (Figure
I, available online at
http://www.atvb.ahajournals.org). In vivo expression of gp19k was
detected by reverse transcriptase–based polymerase chain reaction
(RT-PCR) performed on arterial extracts.
For 2 reasons, we did not determine the affinity of gp19k for rabbit
major histocompatibility class (MHC) I. First, these experiments might
not be predictive of in vivo function, as gp19k appears to have
immunomodulatory activity that is not explained by its affinity for
MHC-I.13 15 Second, the ability of gp19k to improve vector
performance is most definitively tested in vivo by direct
measurement of the critical end points of duration of expression and
magnitude of host immune response. We detected expression of the
E2A-encoded DNA-binding protein16 by Western blotting of
cell extracts by using antibodies provided by Dr G. Schouten
(IntroGene; Figure II, available online at
http://www.atvb.ahajournals.org).
Animal Experiments
Vector stocks (
75 µL per artery) were infused in the lumen
of surgically isolated common carotid arteries of adult male, specific
pathogen–free, New Zealand White rabbits (Charles River Laboratories,
Montreal, Quebec, Canada; 3.0 to 3.5 kg) as previously
described.17 Arteries were harvested from 1 hour to 14
days later and processed for the ß-galactosidase (ß-gal) activity
assay, histological analysis, or DNA
extraction.
AdRSVnLacZ (2 independent preparations), Adgp19kLacZ (1 preparation), and AdCMVNull (2 independent preparations) were infused at 4 to 5x109 plaque-forming units (pfu)/mL (2 to 6x1011 particles/mL). Three independent preparations of AdE2AtsLacZ were required to complete the experiments. These preparations were infused either at 5x109 LacZ-transducing units/mL or at 5x109 relative pfu/mL, derived from comparison with AdRSVnLacZ.
Central venous cannulas were placed in rabbits assigned to receive either CTX or control saline infusions. CTX (30 mg · kg-1 · d-1, Sigma) or sterile saline was administered by catheter for 4 days preoperatively and 14 days postoperatively.
ß-Gal Activity Assay
ß-Gal activity in lysates of arteries was measured with a
chemiluminescence assay.1
Southern Blot Analysis
DNA was extracted from cultured cells or carotid artery segments
by SDS–proteinase K digestion. Blots were hybridized with a
32P-labeled, 2.9-kb HindIII fragment
of adenovirus 5 (a probe for the E4 region). To permit quantification
of adenoviral DNA in transduced tissues, each autoradiograph included a
standard curve generated by electrophoresis and blotting of purified,
HindIII-digested adenoviral DNA loaded in amounts
corresponding to 0.1 to 300 copies per haploid genome (ie, 0.2 to 600
copies per diploid cell) in the experimental lanes. Radioactive signals
on blots were quantified with a PhosphorImager.
Real-Time PCR Quantification of Adenoviral DNA
Adenoviral DNA was also quantified by real-time "Taq-Man"
quantitative PCR, by using amplification and detection protocols
similar to those described elsewhere.18 To aid in
quantification of adenoviral DNA in experimental samples, each
amplification included a standard curve in which wells were spiked with
102 to 108 copies of
adenoviral DNA.
Detection of Anti-Adenoviral Antibodies
Serum antibodies to adenovirus were measured by ELISA. In brief,
96-well plates were coated with 100 µL of adenoviral vector stock.
The wells were rinsed and blocked, and 100 µL of diluted rabbit serum
was added to each well. Bound antibody was detected with alkaline
phosphatase–conjugated mouse anti-rabbit IgG.
Histological Analysis
Arteries were embedded and sectioned, and serial sections were
stained for CD5 (a T-cell antigen), vascular cell adhesion molecule-1
(VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) expression, as
described.2 The mean score of the 4 sections from each
artery was calculated and assigned as an overall score for the artery.
Individual artery scores were grouped according to treatment, and the
treatment groups were compared by using the individual artery scores as
experimental units.
Statistical Analysis
The effect of CTX on persistence of DNA was evaluated by
unpaired t test; these data are presented as
mean±SEM. Data not normally distributed (or for which variances of
experimental groups were unequal) were compared by rank-sum testing (2
groups) or Kruskal-Wallis ANOVA (3 groups). The strength of
correlations was assessed by Spearman’s rank-order test, and the
variability of hematological values over time was evaluated by 2-way
repeated-measures ANOVA with Tukey’s test.19
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In Vivo ß-Gal Expression Is Not Prolonged by Second-Generation Vectors
We infused AdRSVnLacZ, AdE2AtsLacZ, and Adgp19kLacZ into rabbit common carotid arteries, harvested the arteries 3 and 14 days later, and measured ß-gal activity in arterial extracts. Three days after gene transfer, arteries infused with the 3 vectors had similar levels of ß-gal expression (Figure 2
, P=0.2). At 14 days,
as expected, ß-gal expression in AdRSVnLacZ-transduced arteries
declined by >98% (P=0.02 versus 3 days). ß-Gal
expression also declined significantly at 14 days in arteries
transduced with the second-generation vectors (P<0.03
versus 3 days for both AdE2AtsLacZ and
Adgp19kLacZ). Moreover, expression levels at 14 days were equivalent
for all 3 vectors (P=0.46).
|
Systemic Immunosuppression Only Marginally Prolongs ß-Gal
Transgene Expression
The initial results with AdE2AtsLacZ and
Adgp19kLacZ suggested that circumventing the immune responses to
adenoviral proteins by vector engineering might not be an effective
strategy for prolonging transgene expression. To test whether direct
suppression of the immune response to adenovirus can prolong transgene
expression in this model, we measured ß-gal expression in 14-day
arteries from rabbits infused with AdRSVnLacZ and treated with CTX.
Treatment for 18 days with CTX (from 4 days before gene transfer until
harvest) effectively immunosuppressed the rabbits (the Table
and see below) and caused relative
preservation of ß-gal transgene expression (P=0.04 versus
14-day AdRSVnLacZ arteries without CTX; Figure 2). Nonetheless,
immunosuppression did not prevent a significant decline in ß-gal
activity in AdRSVnLacZ arteries between 3 and 14 days (94% drop,
P=0.05).
|
Vascular Inflammation and the Humoral Immune Response to Adenovirus
Are Not Decreased by Second-Generation Vectors
To test the hypothesis that these second-generation adenoviral
vectors produce less vascular inflammation, we measured T-cell
infiltration and expression of ICAM-1 and VCAM-1 14 days after infusion
of AdE2AtsLacZ, Adgp19kLacZ, or AdRSVnLacZ (both
with and without CTX treatment). As expected, arteries infused with
AdRSVnLacZ had substantial T-cell infiltration and expression of ICAM-1
and VCAM-1 (Figure 3). CTX essentially
eliminated T-cell infiltrates and VCAM-1 expression (P=0.001
and 0.02, respectively, versus AdRSVnLacZ without CTX) but did not
significantly alter ICAM-1 expression (P=0.16). Neither of
the second-generation vectors produced significantly less inflammation
than the first-generation vector AdRSVnLacZ, although there was a trend
toward less T-cell infiltration and ICAM-1 expression in arteries
transduced with Adgp19kLacZ (P=0.07 for both measurements
versus AdRSVnLacZ without CTX). Because the second-generation vectors
produced fewer antigens (AdE2AtsLacZ) or
potentially decreased antigen presentation (Adgp19kLacZ),
we considered the possibility that the humoral immune response to these
vectors might be less. Serum anti-adenoviral IgG was measured in
naïve rabbits (no known exposure to human adenovirus) and in
rabbits 14 days after carotid infusion of 1 of the 3 vectors. As
expected, infusion of AdRSVnLacZ provoked a robust, humoral immune
response (Figure 4). This response was
eliminated in rabbits treated with CTX (P<0.005 versus
AdRSVnLacZ without CTX). However, anti-adenoviral IgG levels did not
differ between rabbits infused with AdE2AtsLacZ
or Adgp19kLacZ and rabbits infused with AdRSVnLacZ
(P=0.89).
|
|
Adenoviral Vector DNA Is Rapidly Lost From the Arterial
Wall
The substantial and significant decline of ß-gal transgene
expression, despite CTX treatment, suggested that loss of transgene
expression was largely independent of specific immune responses to
adenoviral antigens. Two alternative mechanisms for loss of transgene
expression required consideration. First, ß-gal expression might be
lost due to a specific immune response to ß-gal protein, leading to
elimination of ß-gal–expressing vectors solely as a result of the
specific elimination of ß-gal–expressing cells. Second, ß-gal
expression might be lost due to transcriptional shutdown despite
persistence of vector DNA. To address the 2 possibilities
simultaneously, we infused AdCMVNull (a first-generation
vector that does not express a marker protein) and followed vector DNA
persistence by quantitative Southern blotting and real-time PCR.
Removing the ß-gal transgene and measuring vector DNA instead of
transgene expression would reveal both whether transgene immunogenicity
was the critical factor leading to loss of transgene
expression5 and whether vector DNA persisted despite loss
of expression in this model.20
Arteries transduced with AdCMVNull at
4x109 pfu/mL and harvested 1 hour later
contained a large amount of vector DNA: 350 copies per
arterial wall cell (Figure 5A). Vector DNA declined rapidly, with
<0.5% of the initial amount of vector DNA present by 14 days. To
determine whether the rate of vector DNA loss was dose related (as
could occur if high concentrations of vector were directly cytotoxic),
we repeated the time-course experiment at 2 lower doses:
4x108 and 1x108 pfu/mL.
Vector DNA delivered at all 3 doses was lost from the
arterial wall with identical kinetics: simple exponential
decay with a half-life of 1 day (Figures 5B and 5C).
|
We next determined whether CTX immunosuppression prevented the loss of
vector DNA. Twenty arteries (in 10 rabbits) were infused with AdCMVNull
at 4x108 pfu/mL. Beginning 4 days before gene
transfer, 5 of the rabbits were treated with daily CTX and 5 were
treated with saline, as described above. At 3 days after gene transfer,
arteries from both groups had equivalent amounts of vector DNA
(4.6±1.7 versus 3.0±1.8 copies per arterial wall cell,
Figure 5D; P=0.48). At 7 days, arteries from
CTX-treated rabbits had 7-fold more vector DNA than did arteries from
saline-treated rabbits (0.58±0.16 versus 0.082±0.014 copies per
arterial wall cell, P=0.036). Thus, CTX
immunosuppression slowed the loss of viral DNA, but only modestly.
Despite CTX treatment, nearly 90% of vector DNA present on day 3
is lost by day 7. These results paralleled the effects of CTX
treatment on ß-gal expression (Figure 2). Taken together, the
data suggest that antigen-specific immune responses to adenoviral and
transgene proteins play, at most, a minor role in the early loss of
vector DNA and transgene expression.
Finally, to ensure that our data on vector DNA persistence were
not specific to vectors generated in our laboratory, we repeated DNA
persistence studies with the null vectors AdE2A+ and AdE2A-. Both
vectors were infused at doses of 1 to 2x109
IU/mL, and arteries were harvested either 1 or 12 days after infusion.
Both vectors were lost from the arterial wall with a time
course similar to that of AdCMVNull (Figure 5E).
Adenovirus-Induced Vascular Inflammation Is Dose Dependent and
Occurs Independently of Vector Loss or E2A Expression
CTX immunosuppression nearly eliminated arterial wall
inflammation but only modestly extended the persistence of ß-gal
transgene expression and AdCMVNull DNA (Figures 2 and 5D). These data suggested that loss of vector DNA from the
arterial wall was largely independent of a local
inflammatory response. To test whether the dissociation between
inflammation and vector loss was restricted to experiments performed
with vectors generated in our own laboratory, we performed additional
experiments with the first- and second-generation null viruses AdE2A+
and AdE2A-. The vectors were infused at 2 doses: 1 to
2x109 IU/mL and 1 to 2x1010 IU/mL.
In arteries infused with 1 to 2x109 IU/mL of
either vector, inflammation was nearly undetectable (Figure 6). Thus, rapid loss of both vector
genomes from the artery wall (Figure 5E) is not dependent on a
robust local inflammatory response. At 10-fold higher doses, both
vectors caused significantly more inflammation, with no significant
differences between AdE2A+ and AdE2A- (P>0.09 for all 6
comparisons between the vectors). Moreover, arteries infused with
AdCMVNull at 4x108 pfu/mL (equivalent by
Southern titration to 1 to 2x109 IU/mL of AdE2A+
and AdE2A-) also exhibited only minimal vascular inflammation (data
not shown), despite rapid loss of vector DNA (Figures 5B and 5C). Thus, for all vectors tested, infusion at lower doses
significantly decreased inflammation but did not increase the
persistence of vector DNA.
|
P=0.065) different between
groups indicated. |
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We tested whether second-generation adenoviral vectors would prolong recombinant gene expression or decrease inflammation after arterial gene transfer. Our major findings were as follows: (1) neither of 2 ß-gal–expressing, second-generation vectors prolonged transgene expression, decreased the humoral immune response to adenovirus, or produced significantly less local inflammation; (2) viral DNA is rapidly lost from the arterial wall, with kinetics that parallel loss of transgene expression from the ß-gal–expressing vectors; (3) essentially complete elimination of the humoral and tissue immune responses to adenovirus by CTX had relatively minor effects on the duration of transgene expression or persistence of adenoviral DNA; and (4) arterial wall inflammation after adenovirus infusion is dose dependent and does not require expression of an immunogenic transgene. Taken together, our results suggest that new paradigms are required to explain the loss of adenovirus-mediated gene expression from the artery wall and to design strategies to prevent this loss.
In contrast to previous reports,7 21 22 23 inclusion of the temperature-sensitive E2A mutation did not affect either inflammation or the persistence of transgene expression. There are several potential explanations for this discrepancy. First, reversion to wild-type virus is possible. However, this is an unlikely explanation, because the E2A mutation has a low rate of spontaneous reversion14 and all of our in vivo experiments were performed with low-passage virus. Second, the preparations could have been contaminated with E1A+ virus, generated by recombination in the 293 cells. However, it is unlikely that occult E1A positivity confounded our experiments because the sensitivity of the PCR assay is such that few, if any, rabbit cells could have been transduced with an E1A+ virus. Third, animal strain and species differences can cause discrepant results in adenoviral gene transfer experiments.24 One group reported prolonged expression and decreased inflammation with E2A temperature-sensitive viruses in CBA mice, cotton rats, and rhesus monkeys.21 22 23 Another group found no advantage of the same E2A mutation in BALB/c mice and dogs.25 The absence of an effect in rabbits suggests that the applicability of this strategy is limited. Fourth, the negative results with AdE2AtsLacZ might be attributable to a destructive immune response to the ß-gal gene product, which is a potent antigen.26 27 However, this is an extremely unlikely explanation. The ß-gal reporter gene was also used in studies in which the E2A mutation prolonged gene expression,7 21 22 whereas expression of a species-homologous cDNA (eg, canine factor IX in dogs) was not prolonged by the E2A mutation.25 In addition, DNA from AdCMVNull, AdE2A+, and AdE2A- (none of which express ß-gal) was rapidly lost from the arterial wall.
Because MHC-I–restricted immune responses may play a major role in the inflammation leading to elimination of adenovirus-transduced cells,4 28 MHC-I molecules are logical targets for strategies designed to improve adenovirus-mediated gene transfer. Adenovirus gp19k protein inhibits MHC-I function by retaining MHC-I molecules in the endoplasmic reticulum.13 Indeed, the ability of adenovirus to establish persistent infections29 has been attributed, at least in part, to the properties of gp19k.30 Moreover, gp19k may have immunomodulatory properties that are independent of its ability to bind MHC-I.13 15
Three previous studies have examined whether expression of gp19k could
improve adenoviral vector performance. One of those studies
reported positive results,8 whereas another failed to find
a beneficial effect of gp19k expression.31 A third study
yielded strain-specific results.32 In the present
study, we found no effect of gp19k expression on the persistence of
transgene expression or the humoral immune response. Potential
explanations for these observations include the inability of gp19k to
bind rabbit MHC-I alleles, unfavorable kinetics of gp19k expression
(ie, expression after upregulation of MHC-I),30 and
presentation of antigens after phagocytosis (which would
uncouple gp19k expression from antigen
presentation).33 The tendency toward decreased
inflammation in Adgp19kLacZ-transduced arteries raises the intriguing
prospect that gp19k may have an anti-inflammatory effect, at least in a
subset of rabbits (Figure 3; note the bimodal distribution of
data points in the Adgp19kLacZ groups). However, this possibility may
not be terribly important in the absence of any effect of gp19k on the
duration of transgene expression.
CTX blocked the humoral immune response to adenovirus and nearly
eliminated T cells from the arterial wall. Although CTX
increased ß-gal expression at 14 days compared with first- or
second-generation vectors, expression nevertheless dropped
substantially (94%) below 3-day levels (Figure 2). These data
suggest either that CTX-insensitive immune responses are responsible
for loss of transgene expression or that expression declines because of
nonimmune mechanisms. It will be difficult to discriminate between
these possibilities in a rabbit model, in which strains with targeted
deletions of immune system components are not available.
We recently reported that ß-gal transgene expression is also rapidly
lost (ie, 90% drop in ß-gal expression between 3 and 14 days) after
infusion of AdRSVnLacZ to carotid arteries of mice that lack
antigen-specific immunity.11 We attributed the loss of
transgene expression in mouse carotid arteries to elevated cell
turnover, occurring largely as a result of surgical manipulation. We
were concerned, however, that stimulation of cell turnover and loss of
gene expression might be specific to mouse arteries, which are easily
damaged during surgery. The present study (especially the results
obtained with CTX) confirms the dissociation between effective
immunosuppression and persistence of transgene expression after
arterial gene transfer. Moreover, the present study
also shows that vector DNA is lost from the artery wall beginning on
the first day after vector infusion and continuing with the same
kinetics (half-life of 1 day) for at least a week. If vector
persistence were primarily controlled by antigen-specific immune
responses, then vector DNA would be expected to persist for a few days
and then decline rapidly. Alternatively, if early
macrophage-mediated clearance of vector DNA were followed by
antigen-specific clearance of transduced cells, elimination of
adenoviral DNA would be biphasic.34 Neither of these
models fits the observations that we have made in arterial
gene transfer models. Instead, the dissociation of immune responses
from transgene persistence contrasts with numerous studies that
correlate persistence of adenovirus-mediated transgene expression with
genetic deficiencies in the antigen-specific immune
system.4 6 24 27 28 None of these other gene transfer
studies, however, were performed in arteries.
There are at least 2 aspects of our results that bode well for the
future of adenovirus-mediated arterial gene transfer.
First, the amount of vector DNA present in the artery wall
immediately after vector infusion is surprisingly high. At the highest
dose infused (4x109 pfu/mL), there were >300
copies of vector DNA per arterial wall cell 1 hour after
infusion, and >100 copies remained after 1 day. Even at the lowest
dose tested (1x108 pfu/mL), 4 vector copies per
artery wall cell were present at 1 day. If, as suggested by the
pattern of reporter gene expression in this model,2 17 35
virtually all vector copies are in the endothelium,
then the number of vector copies per target cell is far higher. If all
of these copies are intracellular, then efficient gene delivery to the
endothelium (=1 copy per cell) might be achieved at
surprisingly low vector doses. The second positive aspect of our
results is that artery wall inflammation can be nearly eliminated by
lowering the dose of virus (Figure 6). Thus, optimal vector
design may allow high levels of transgene expression at vector doses
that do not cause local inflammation.
In summary, factors that control the early loss of arterial wall transgene expression appear to differ from factors that govern adenovirus-mediated transgene loss in other tissues. Identification of these factors, which may include cell turnover,11 instability of adenoviral DNA, and noncytotoxic pathways of virus elimination from host cells,36 is essential for developing strategies that extend the utility of adenovirus-mediated arterial gene transfer.
|
Acknowledgments |
|---|
This work was supported in part by the Daiichi Pharmaceutical Company, by IntroGene, and by grants to the coauthors: Dr Dichek (HL 60504), Dr Schneider (Pacific Vascular Research Foundation), and Dr Vassalli (Swiss National Science Foundation, Swiss Biomedical Foundation, and Swiss Association Against Hypertension). Dr Dichek is an Established Investigator of the American Heart Association. We thank R. Linnemann for technical assistance; Dr W. Wold for a gp19k-expressing plasmid vector used as a positive control; Dr J. Yewdell for the Tw1.3 antibody; Dr G. Schouten for AdE2A+ and AdE2A-, as well as antisera to E2A DNA-binding protein; and Dr M. Kay for helpful discussions. Facilities for kinetic PCR assays were supported by the UCSF Center for AIDS Research (No. P30 MH590437) and the UCSF/Macy’s Creative Therapies Program. N. Shea and J. Carroll are acknowledged for graphics assistance, G. Howard and S. Ordway for editorial assistance, and S. Gonzalez and C. Goodfellow for photography.
|
Footnotes |
|---|
Shan Wen and Darren B. Schneider contributed equally to this study.
Received January 19, 2000; accepted March 15, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Lee SW, Trapnell BC, Rade JJ, Virmani R, Dichek
DA. In vivo adenoviral vector–mediated gene transfer into
balloon-injured rat carotid arteries. Circ Res. 1993;73:797–807.
[Abstract/Free Full Text] - Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, Libby P, Dichek DA. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia. J Clin Invest. 1995;96:2955–2965.
-
Channon KM, Qian H, Youngblood SA, Olmez E, Shetty GA,
Neplioueva V, Blazing MA, George SE. Acute host-mediated
endothelial injury after adenoviral gene transfer in
normal rabbit arteries: impact on transgene expression and
endothelial function. Circ Res. 1998;82:1253–1262.
[Abstract/Free Full Text] - Yang Y, Li Q, Ertl HCJ, Wilson JM. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol. 1995;69:2004–2015.[Abstract]
- Tripathy SK, Black HB, Goldwasser E, Leiden JM. Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat Med. 1996;2:545–550.[Medline] [Order article via Infotrieve]
-
Dai Y, Schwarz EM, Gu D, Zhang W-W, Sarvetnick N,
Verma IM. Cellular and humoral immune responses to adenoviral vectors
containing factor IX gene: tolerization of factor IX and vector
antigens allows for long-term expression. Proc Natl Acad Sci
U S A. 1995;92:1401–1405.
[Abstract/Free Full Text] -
Engelhardt JF, Ye X, Doranz B, Wilson JM. Ablation of
E2A in recombinant adenoviruses improves transgene
persistence and decreases inflammatory response in mouse liver.
Proc Natl Acad Sci U S A. 1994;91:6196–6200.
[Abstract/Free Full Text] - Lee MG, Abina MA, Haddada H, Perricaudet M. The constitutive expression of the immunomodulatory gp19 k protein in E1–,E3– adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector. Gene Ther. 1995;2:256–262.[Medline] [Order article via Infotrieve]
-
Kochanek S, Clemens PR, Mitani K, Chen H-H, Chan S,
Caskey CT. A new adenoviral vector: replacement of all viral coding
sequences with 28 kb of DNA independently expressing both full-length
dystrophin and ß-galactosidase. Proc Natl Acad Sci
U S A. 1996;93:5731–5736.
[Abstract/Free Full Text] -
Finkel T. Thinking globally, acting locally: the
promise of cardiovascular gene therapy. Circ
Res. 1999;84:1471–1472.
[Free Full Text] - Vassalli G, Agah R, Qiao R, Aguilar C, Dichek DA. A mouse model of arterial gene transfer: antigen-specific immunity is a minor determinant of the early loss of adenovirus-mediated transgene expression. Circ Res. 1999;85:e25–e32.
-
Dong G, Schulick AH, DeYoung MB, Dichek DA.
Identification of a cis-acting sequence in the human
plasminogen activator inhibitor
type-1 gene that mediates transforming growth factor-ß1
responsiveness in endothelium in vivo. J
Biol Chem. 1996;271:29969–29977.
[Abstract/Free Full Text] - Wold WSM, Tollefson AE, Hermiston TW. E3 transcription unit of adenovirus. Curr Top Microbiol Immunol. 1995;199:237–274.
-
Ensinger MJ, Ginsberg HS. Selection and preliminary
characterization of temperature-sensitive mutants of type 5 adenovirus.
J Virol. 1972;10:328–339.
[Abstract/Free Full Text] -
Ginsberg HS, Lundholm-Beauchamp U, Horswood RL, Pernis
B, Wold WSM, Chanock RM, Prince GA. Role of early region 3 (E3) in
pathogenesis of adenovirus disease. Proc Natl Acad Sci
U S A. 1989;86:3823–3827.
[Abstract/Free Full Text] -
Kruijer W, van Schaik FMA, Sussenbach JS. Structure and
organization of the gene coding for the DNA binding protein of
adenovirus type 5. Nucleic Acids Res. 1981;9:4439–4457.
[Abstract/Free Full Text] -
Schneider DB, Vassalli G, Wen S, Driscoll RM, Sassani
AB, DeYoung MB, Linnemann R, Virmani R, Dichek DA. Expression of Fas
ligand in arteries of hypercholesterolemic rabbits
accelerates atherosclerotic lesion formation. Arterioscler Thromb
Vasc Biol.. 2000;20:298–308.
[Abstract/Free Full Text] - Becker K, Pan D, Whitley CB. Real-time quantitative polymerase chain reaction to assess gene transfer. Hum Gene Ther. 1999;10:2559–2566.[Medline] [Order article via Infotrieve]
- Zar JH. Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall; 1984.
- Chen H-H, Mack LM, Choi S-Y, Ontell M, Kochanek S, Clemens PR. DNA from both high-capacity and first-generation adenoviral vectors remains intact in skeletal muscle. Hum Gene Ther. 1999;10:365–373.[Medline] [Order article via Infotrieve]
- Yang Y, Nunes FA, Berencsi K, Gönczöl E, Engelhardt JF, Wilson JM. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994;7:362–369.[Medline] [Order article via Infotrieve]
- Engelhardt JF, Litzky L, Wilson JM. Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a. Hum Gene Ther. 1994;5:1217–1229.[Medline] [Order article via Infotrieve]
- Goldman MJ, Litzky LA, Engelhardt JF, Wilson JM. Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study. Hum Gene Ther. 1995;6:839–851.[Medline] [Order article via Infotrieve]
- Barr D, Tubb J, Ferguson D, Scaria A, Lieber A, Wilson C, Perkins J, Kay MA. Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient inbred strains. Gene Ther. 1995;2:151–155.[Medline] [Order article via Infotrieve]
- Fang B, Wang H, Gordon G, Bellinger DA, Read MS, Brinkhous KM, Woo SLC, Eisensmith RC. Lack of persistence of E1– recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs. Gene Ther. 1996;3:217–222.[Medline] [Order article via Infotrieve]
- Roessler BJ, Allen ED, Wilson JM, Hartman JW, Davidson BL. Adenoviral-mediated gene transfer to rabbit synovium in vivo. J Clin Invest. 1993;92:1085–1092.
- Michou AI, Santoro L, Christ M, Julliard V, Pavirani A, Mehtali M. Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Ther. 1997;4:473–482.[Medline] [Order article via Infotrieve]
- Yang Y, Ertl HCJ, Wilson JM. MHC class I–restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity. 1994;1:433–442.[Medline] [Order article via Infotrieve]
- Wadsworth SC, Zhou H, Smith AE, Kaplan JM. Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo. J Virol. 1997;71:5189–5196.[Abstract]
- Routes JM, Cook JL. Resistance of human cells to the adenovirus E3 effect on class I MHC antigen expression: implications for viral immunity. J Immunol. 1990;144:2763–2770.[Abstract]
- Schowalter DB, Tubb JC, Liu M, Wilson CB, Kay MA. Heterologous expression of adenovirus E3-gp19K in an E1a deleted adenovirus vector inhibits MHC-I expression in vitro, but does not prolong transgene expression in vivo. Gene Ther. 1997;4:351–360.[Medline] [Order article via Infotrieve]
- Bruder JT, Jie T, McVey DL, Kovesdi I. Expression of gp19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver. J Virol. 1997;71:7623–7628.[Abstract]
- Rawle FC, Tollefson AE, Wold WSM, Gooding LR. Mouse anti-adenovirus cytotoxic T lymphocytes: inhibition of lysis by E3 gp19K but not E3 14.7K. J Immunol. 1989;143:2031–2037.[Abstract]
- Worgall S, Wolff G, Falck-Pedersen E, Crystal RG. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum Gene Ther. 1997;8:37–44.[Medline] [Order article via Infotrieve]
- Schneider DB, Sassani AB, Vassalli G, Driscoll RM, Dichek DA. Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors. J Vasc Surg. 1999;29:543–550.[Medline] [Order article via Infotrieve]
- Guidotti LG, Chisari FV. Cytokine-induced viral purging: role in viral pathogenesis. Curr Opin Microbiol. 1999;2:388–391.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  A. S. Narang and R. I. Mahato Biological and biomaterial approaches for improved islet transplantation. Pharmacol. Rev., June 1, 2006; 58(2): 194 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Otsuka, R. Agah, A. D. Frutkin, T. N. Wight, and D. A. Dichek Transforming Growth Factor Beta 1 Induces Neointima Formation Through Plasminogen Activator Inhibitor-1-Dependent Pathways Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 737 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wen, S. Graf, P. G. Massey, and D. A. Dichek Improved Vascular Gene Transfer With a Helper-Dependent Adenoviral Vector Circulation, September 14, 2004; 110(11): 1484 - 1491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Falkenberg, C. Tom, M. B. DeYoung, S. Wen, R. Linnemann, and D. A. Dichek Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth PNAS, August 6, 2002; 99(16): 10665 - 10670. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wen, R. M. Driscoll, D. B. Schneider, and D. A. Dichek Inclusion of the E3 Region in an Adenoviral Vector Decreases Inflammation and Neointima Formation After Arterial Gene Transfer Arterioscler. Thromb. Vasc. Biol., November 1, 2001; 21(11): 1777 - 1782. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||