Published online before print April 27, 2001, doi: 10.1161/hh0901.090926
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2001 American Heart Association, Inc.
Molecular Medicine |
Improved Adenoviral Vector for Vascular Gene Therapy
Beneficial Effects on Vascular Function and Inflammation
From Duke University Medical Center (H.S.Q., V.N., S.E.G.), Durham, NC; Department of Cardiovascular Medicine (K.C.), John Radcliffe Hospital, Oxford, UK; and Cell Genesys Inc (Q.W., M.F., L.T., J.M.), Foster City, Calif.
Correspondence to James McArthur, Cell Genesys Inc, Department of Preclinical Biology and Immunology, 344 Lakeside Dr, Foster City, CA 94404. E-mail to jamesm{at}cellgenesys.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—First-generation, E1-deleted adenoviral vectors (
E1-AV) can transduce the vascular
endothelium with high efficiency, but their use is
limited by the resulting acute endothelial injury and
the long-term development of intimal hyperplasia. To reduce the impact
of viral proteins on the gene-modified cells, a second-generation
adenoviral vector with an additional pair of deletions in the E4 region
was developed. To determine whether this E1/E4-AV vector
would be useful for vascular gene transfer, we directly compared the
efficiency of gene transfer to uninjured rabbit carotid arteries using
either an E1/E4-AV or an E1-AV vector encoding
ß-galactosidase. Both vectors efficiently transduced vascular
endothelium; however, the E1/E4-AV vector
gene–modified vessels showed higher ß-galactosidase expression 10
days after gene transfer. Importantly, the E1/E4-AV vector
produced substantially less endothelial cell
activation, less inflammation, and reduced neointimal
hyperplasia compared with the E1-AV vector–treated vessels. The
E1-AV vector–transduced vessels also demonstrated significantly
impaired endothelium-dependent relaxation whereas the
E1/E4-AV vector did not impact vasomotor function, even at doses
of virus in 5-fold excess of the amount required for >90%
transduction of the endothelium. We conclude that the
E1/E4-AV vector is superior to the E1-AV vector for vascular
gene therapy because of the prolonged transgene expression, reduced
vascular inflammation, reduced intimal hyperplasia, and
maintenance of normal vasomotor function.
Key Words: vascular gene therapy • adenoviral vectors
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recombinant E1-deleted adenoviral vectors (
E1-AV) have proven to be a powerful
tool for vascular gene transfer and shows promise for gene therapy of
restenosis,1 2 3 4 5 6 7 8 vascular thrombosis, and
vein graft
failure. 10–16 However, adenoviral gene transfer may be limited by vector-induced
acute and chronic inflammatory responses that injure the
endothelium and promote intimal
hyperplasia.17 18 The leaky expression of early genes in the first-generation E1-AV
allows low-level transcription of viral late genes and expression of
viral proteins that result in virus-mediated cytopathology, as well as
a virus-specific host immune
response.19 20 21 22 23 24
To address these problems, first-generation E1-deleted
adenoviral vectors have been modified to further reduce adenoviral
protein
expression. 25–30 One such approach combines an E1 deletion with additional deletions in
the E4 region. The E4 region encodes a series of 6 proteins that are
involved with viral DNA replication and viral RNA transport. To this
end, Wang et al 30 created
the E1- and E4-deleted adenoviral vector in which only the E4 orf4
remained. Intravenous administration of equivalent doses of
the E1-AV and E1/E4-AV vectors initially produce similar
levels of transgene expression; however, the mice that receive the
E1/E4-AV vector demonstrate greatly reduced virus-induced
hepatitis and inflammatory cell infiltrates as well as prolonged
transgene
expression.31 32
We undertook the present study to establish whether
vascular gene therapy with the E1/E4-AV vector would avoid the
impairment of vessel function and intimal hyperplasia observed with
first-generation AV vectors. Our results show that the
E1/E4-AV–treated vessels demonstrated prolonged transgene
expression, markedly reduced endothelial activation,
vascular inflammation, and intimal hyperplasia, and had little or no
detrimental impact on endothelial vasomotor function
relative to E1-AV vector–treated vessels. These improved
characteristics represent a significant advance for vascular
applications of adenoviral gene transfer.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Adenovirus
The
E1-AV and E1/E4-AV vectors encoding the
phosphoglyceratekinase (PGK)-promoter ß-galactosidase
(ß-gal) transgene expression cassette were generated as previously
described.30 Both the
E1/E4 and E1 vectors viruses used in the described studies
retain the E3 region genes. The E1/E4-AV vectors were produced in
293 cells that expressed the E4-orf6 gene under the control of a
cAMP-inducible promoter.30
The virus was purified by two rounds of CsCl gradient
centrifugation, dialyzed against PBS containing 10%
glycerol, and stored in PBS containing 10% glycerol and 50 mg/mL human
serum albumin. The frequency of replication-competent
adenovirus has been determined to be
<10–10 in E1-AV preparation and
<10–11 in the E1/E4-AV preparations
of virus (data not shown). Particle titer was determined by using
optical density on a spectrophotometer (Beckman DU640). The particle
titers for the E1-AV and E1/E4-AV preparations were
4x1012 vp/mL and
6x1012 vp/mL, respectively. Infectious
titers were determined by infecting 293 cells with an increasing number
of virus particles and quantifying lacZ expression with a fluorescein
di-ß-D-galactopyranoside (FDG)–based
fluorescence-activated cell sorter assay. The
functional titers for the E1-AV and E1/E4-AV preparations were
1x1011 fp/mL and
6.7x1010 fp/mL, respectively. The total to
functional particle ratios for the preparations of E1-AV and
E1/E4-AV used in these studies were 40:1 and 90:1,
respectively.
Animals and Gene Delivery
Male New Zealand white rabbits (2 to 2.5 kg) were
maintained on a normal diet. Anesthesia was induced with
ketamine and xylazine subcutaneously as previously
described.18 All animal care
and procedures were approved by Duke University Institutional Animal
Care and Use Committee and compiled with the
Guide for the Use and Care of Laboratory
Animals (NIH publication 80-23, revised 1985).
For gene transfer to the carotid artery, a midline incision was made, and the external carotid artery was exposed. The vessel was clamped, and an arteriotomy was performed. Approximately 200 µL of virus solution was infused per vessel. This volume was sufficient to expand the vessel to physiological dimensions. The solution was allowed to dwell for 15 minutes and was removed. The artery was then flushed with Dulbecco’s modified Eagle’s (DME) medium, and the arteriotomy was closed. The clamps were then removed, and circulation was restored. High-titer viral stock was diluted with DME medium/virus storage medium to ensure equal composition of virus solutions at different viral titers. Virus doses given to each artery were 1x109, 5x109, and 2.5xx1010 fp. Sham infections were performed with DME/virus storage medium alone.
Vessel Harvesting and Analysis
Vessels were harvested at time points indicated in
the figures as previously
described.18 Briefly,
animals were anesthetized and heparinized (700 IU
intravenously), and the carotid arteries were dissected
free. Animals were then killed with 100 mg/kg intravenous
pentobarbital. Vessels were immediately excised and washed in PBS.
Segments from each vessel were processed for various analyses.
They were (1) immediately frozen at -80°C for ß-gal
quantification, (2) equilibrated in 30% sucrose and frozen in OCT
(Miles Laboratories) for immunohistochemistry, (3) fixed in 10%
formalin and paraffin embedded for histological stains,
or (4) were hung fresh without delay in organ baths for vasomotor
studies.
Histological Methods
LacZ Staining
Vessels were equilibrated in 30% sucrose in PBS
briefly and then frozen in liquid nitrogen. Snap-frozen vessel segments
were cut into 6-µm sections and stained with
5-bromo-4-chloro-3-indolyl-b-D-galactosidase
(X-gal) solution for 4 hours at room
temperature.18 Total protein
was also extracted and quantified for ß-gal protein by using an ELISA
kit and for total protein by using a Bradford protein assay.
ß-Gal protein was measured in nanograms of ß-gal per milligram of
total vessel protein.
Immunohistochemistry
Briefly, 6-µm frozen sections were cut and dried at
room temperature and then equilibrated in PBS. Blocking solution (1.5%
horse serum in PBS) was applied for 1 hour at room temperature or
overnight at 4°C. Antibodies were diluted in blocking solution at the
manufacturer’s recommended concentration and were applied to tissue
sections for 1 hour. Immunohistochemistry for lymphocytes was performed
by using primary antibodies directed against rabbit CD18 (from Serotec). Vascular cell adhesion molecule-I
(VCAM-1) and intercellular adhesion molecule-I (ICAM-1) staining was
performed with monoclonal antibodies raised against rabbit VCAM-1 and
ICAM- 1 (a generous gift of Dr. M. Cybulsky, Harvard University,
Boston, Mass).
Vessel Morphology
Paraffin-embedded sections were treated with
Voerhoff’s stain or with Masson’s trichrome stain
(Sigma) to visualize the vessel structure.
Neointimal thickness was measured with the NIH
Image software analysis
program.
Image Analyses
For quantification, image analyses was
performed. Stained sections were visualized by using an
Olympus IX70 inverted microscope, and images
were captured by using Adobe Premiere. The immunohistochemical staining
results were quantified by using NIH Image 1.61 software. Two arteries
per animal were exposed to virus in each treatment group at each dose:
1x109 fp (E1-AV n=10 vessels,
E1/E4-AV n=8 vessels), 5x109 fp
(E1-AV n=42 vessels, E1/E4-AV n=48 vessels), and
2.5x1010 fp (E1-AV n=18 vessels,
E1/E4-AV n=18 vessels). Briefly, cross sections for each vessel
were taken and quantified in 4 to 6 different portions of the vessel at
x5 magnification. The neointimal thickness was determined
on trichrome-stained vessel sections and expressed as millimeters per
high power field. Four to 6 points were measured for each portion per
section.
Vasomotor Studies
Studies to assess vasorelaxation after adenoviral
gene delivery was performed as previously
described.18 Briefly, 5-mm
arterial rings were mounted in 30-mL organ baths containing
oxygenated (95% O2, 5%
CO2) Krebs-Henseleit buffer at 37°C. Briefly,
cumulative dose-response curves to phenylephrine
(10-9 to 10-4
mol/L) were established; vessels were then submaximally precontracted
with phenylephrine (usually
3x10-6 mol/L), and
endothelial function was evaluated by vascular
relaxation in response to acetylcholine
(10-8 to 10-4
mol/L). Statistical significance was assessed with
ANOVA.
TaqMan PCR Analyses
Quantification of adenoviral DNA in the rabbit
carotid artery after gene transfer was determined by using TaqMan
polymerase chain reaction (PCR). DNA extraction of the treated vessel
was performed by using the Qiagen tissue DNA extraction kit. Fifty
nanograms of genomic DNA was analyzed with TaqMan PCR by using
adenovirus primers located upstream of the E4 region of adenovirus. The
following primer set was used to produce a 400-bp amplicon: forward
primer, 5'-ACAACTCCAAGTGCATACTC-3' and reverse primer,
5'-CTGATCAGCGTTTGGCTCGA-3'. The probe used was
6FAM-CTGGTCTGGCCACAACTACATTAA-TAMRA (Applied Biosystems). A final
reaction volume of 50 µL consisted of 1x TaqMan Buffer A; 0.4
µmol/L each primer; 50 nmol/L probe; 0.2 mmol/L each dATP, dCTP,
and dGTP; 0.4 mmol/L dUTP; 4.5 mmol/L
MgCl2; 2.5 units of AmpliTaq GOLD polymerase;
and 0.5 units of AmpErase UNG (Applied Biosystems). A plasmid
containing the adenovirus amplicon sequence was diluted in a tRNA
diluent (25 µg/mL) and used as a positive control series at 10 000,
1 000, 100, 10, 1.0, and 0.1 copies per replicate, and these were run
in triplicate. Each reaction was run under the following conditions:
50°C for a 2-minute hold; 95°C for 10-minute hold; then 40 cycles
of 95°C for 15 seconds and 60°C for 1-minute in an ABI PRISM 7700
Sequence Detection System unit (Applied Biosystems). The results were
analyzed with the Sequence Detection System (version 1.6.3)
software’s default settings (baseline 3 to 15; threshold set to 10x
the standard deviation of the baseline). By using the determination
that 71-ng genomic DNA is equivalent to 10 000 cellular genomes, the
results are expressed in adenovirus DNA genome copies per
cell.
Late Gene Expression by PCR
HeLa cells were seeded in 6-cm dishes at a density of
1x106 cells and transduced with E1-AV
and E1/E4-AV vectors encoding lacZ at functional MOI of 10
and were harvested 4 hours, 24 hours, or 48 hours later. RNA was
isolated from cell pellets, and cDNA was generated by reverse
transcriptase (RT)-PCR by using first-strand synthesis with random
hexamer primers (SuperScript kit by Gibco BRL). The following primers
were designed for amplification of the adenovirus L3 and L5 gene
transcripts: 5'-GGACAATTTTATT-GGTCTAA-3' (L3 forward);
5'-TGTTTGGGTTATCAG-AAATT-3' (L3 reverse),
5'-GAGGACTAAGGATTGATT-3' (L5 forward), 5'-CGTGAGATTTTGGATAAG-3' (L5
reverse). The PCR reactions were set up with the SuperScript kit with
the following conditions: 94°C for 5 minutes, 40 cycles of 94°C for
45 seconds, 60°C for 45 seconds, 72°C for 1 minute, and 72°C for
10 minutes. The PCR products were run out on a 2% agarose gel for
analysis.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
El/E4-AV–ß-Gal–Transduced Rabbit
Carotid Arteries Demonstrate Improved Transgene ExpressionExposure of uninjured rabbit carotid arteries to 5x109 fp of
E1-AV or E1/E4-AV
vector encoding lacZ efficiently transduced the intima with >80% of
the endothelial cells expressing ß-gal 3 days after
gene transfer
(Figure 1a
through 4d). To further quantify ß-gal
expression, an ELISA was performed on whole-vessel lysates 3, 10, and
28 days after gene transfer
(Figure 1e). No significant difference in gene expression was
observed at 3 days between the two vectors, which confirmed that the
titers and infection conditions were essentially equivalent between the
E1-AV and E1/E4-AV vectors. The number of ß-gal–positive
endothelial cells declined considerably by 10 days
after gene transfer; however, there was a clear histologic difference
between E1-AV– and E1/E4-AV–treated arteries with greater
staining in the latter. ß-Gal protein ELISA measurements of the
transduced arteries indicated that ß-gal expression was 4-fold higher
in the E1/E4-AV–treated vessels at the interim time point of 10
days
(Figure 1e). By day 28, ß-gal staining and protein
expression were negligible in vessels treated with either AV vector
(<1%) (data not shown). This decline in protein expression was
paralleled by a decrease in the number of vector genomes
(Figure 2). Maximal genome copy numbers for
E1-AV–transduced vessels were observed at day 3 with a significant
loss in genomes observed 10 and 28 days after gene transfer. The number
of vector genomes was approximately 4-fold higher in
E1/E4-AV–transduced vessels than E1-AV–transduced vessels at
days 10 and 28, consistent with the prolonged ß-gal protein
expression determined by histology and ELISA.
|
) or 
) vector. Vessels were harvested at 3 or 10 days after gene transfer as indicated, and arterial sections were assayed for ß-gal expression levels by using ELISA. Statistically significant differences between
|
Endothelial Activation and
Vascular Inflammation
To compare the degree of endothelial
activation induced by the two vectors, arterial
cryosections were compared for ICAM and VCAM expression by using
immunohistochemistry
(Figure 3). Clear differences in these markers were
observable between sham-treated, E1/E4-AV–treated, and
E1-AV–treated arteries 3, 10, and 28 days after gene transfer. To
quantify the observed differences, image analysis was
performed. This revealed that ICAM and VCAM expression was
significantly higher in the E1-AV–treated arteries compared with
E1/E4-AV–treated arteries. These differences were statistically
significant (P<0.01) and were
observed at all 3 virus doses.
|
) were present in
Recombinant first-generation adenoviral vectors have
been shown to induce the infiltration of neutrophils, lymphocytes, and
monocytes.18 Cellular
infiltration into the E1/E4-AV–transduced vessels was assessed
by using immunohistochemistry with CD18, a marker for neutrophils and
monocytes
(Figure 4a through 4c), and CD43, a pan T-lymphocyte marker
(Figure 4d through 4f). Substantially fewer CD18 and CD43
positive cells were present in E1/E4-AV–treated arteries,
relative to E1-AV. This difference was quantified by both cell
counting (data not shown) and image analysis
(Figure 4g and 4h). As was the case for adhesion molecule
expression, the E1/E4-AV vector induced greater inflammatory cell
infiltration than did the sham treatment but significantly and
substantially less than that observed with the E1-AV
vector.
|
Studies of Vasomotor Function
Exposure of vessels to E1-AV vectors has been shown
to impair endothelium-dependent relaxation in a
titer-dependent manner.18
Vasomotor function of E1/E4-AV vector–treated carotid arteries
was evaluated in organ baths at 3, 10, and 28 days after gene transfer
(Figure 5). The E1-AV vector significantly impaired
endothelium-dependent relaxation at all time points
(Figure 5). In contrast, the E1/E4-AV had surprisingly
little impact on endothelium-dependent relaxation.
Phenylephrine-induced contraction and sodium
nitroprusside–induced relaxation were the same in all vessels,
regardless of treatment (data not shown).
|
) vector– or sham-treated (
) were harvested was evaluated at 3 (a), 10 (b), at 28 (c) days after gene transfer. Vasomotor function was then assessed by precontracting arteries to 90% of maximal tension with phenylephrine and then adding the indicated concentrations of acetylcholine. Untreated vessels (
) are shown as a control. Data are presented as mean % contracted±SE; *P<0.05 versus sham control; **P<0.01 versus sham; +P<0.05 versus
Intimal Thickening
In addition to inducing endothelial
cell activation, inflammatory cell infiltrates, and vessel dysfunction,
the first-generation E1-AV provoked a profound thickening of the
intima. In vessels receiving the 5x109-fp
E1-AV, intimal thickening was readily apparent as early as 10 days
after gene transfer
(Figure 6f). This intimal thickening progressively increased
at day 28 after transduction
(Figure 6h and 6j). A similar but less robust intimal
thickening was observed at the lower dose of E1-AV of
1x109 fp
(Figure 6b, 6d, and 6i). The E1/E4-AV–treated vessels
demonstrated significantly less neointimal thickening at
all doses and time points examined compared with their
E1-AV–treated counterparts
(Figure 6a, 6c, 6e, 6g, 6i, and 6j). As expected, with
increasing doses, E1/E4-AV produced more intimal thickening.
However, even at the highest dose, the E1/E4-AV induced less
neointimal thickening than 5-fold lower doses of the
E1-AV.
|
Reduced Adenovirus Late Gene Expression in the
E1/E4-AV–ß-Gal Vector
These results demonstrate that
E1/E4-AV–ß-gal–transduced vessels experienced reduced vector
associated pathology than the E1-AV–ß-gal–treated vessels. Leaky
expression of adenoviral late genes has been shown to be responsible
for stimulating an immune response against the transduced cells and
inducing cell death of the transduced cells. Indeed, we have observed
that the E1/E4-AV–ß-gal vector late gene expression is reduced
in ex vivo cell cultures. HeLa cells were transduced with either the
E1/E4-AV–ß-gal or E1-AV–ß-gal vectors or wild-type
adenovirus
(Figure 7) and the presence of L3 or L5 late gene transcripts
examined by using RT-PCR 4, 24, and 48 hours later. The ethidium
bromide–stained gels of the PCR products indicated that the L3
transcripts were reduced in both the E1-AV–ß-gal and
E1/E4-AV–ß-gal transduced cells compared with wild-type
adenovirus-infected cells. These gels were then Southern blotted and
probed with a probe specific to the L3 hexon gene. L3 gene expression
was detected in the Southern blot analysis in the
E1-AV–ß-gal vector–transduced cells
(Figure 7, bottom panel) but was still undetectable in the
E1/E4-AV–ß-gal–transduced cells. L5 gene expression was
reduced in the E1/E4-AV–ß-gal–transduced cells compared with
both E1-AV–ß-gal vectors and wild-type adenovirus-transduced
cells. Reduced late gene expression in E1/E4-AV–transduced
vessels may be responsible for the greater persistence of vector
genomes and ß-gal protein over time and the lower levels of
inflammation and intimal thickening observed in the E1/E4-AV
vector–transduced vessels.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
First-generation E1 gene–deleted adenoviral vectors lack essential immediate-early genes–encoding transcription factors that are required for viral DNA replication and the expression of viral late proteins. Although these deletions render the recombinant adenovirus replication incompetent and greatly reduce its cytopathogenicity, transgene persistence is limited by virus- and immune-mediated cytopathology resulting from residual low-level expression of the viral late gene products. Viral late gene expression is reduced in the
E1E4-AV vectors.
Transduction of rabbit carotid arteries with an
E1/E4-AV vector encoding lacZ produced superior persistence of
ß-gal expression and a significant reduction in vessel inflammation,
intimal hyperplasia, and vessel dysfunction compared with the
corresponding E1-AV vector. We have previously shown that
first-generation adenovirus induces titer-dependent impairment in
endothelium-dependent relaxation. The functional
impairment was likely a result of the host inflammatory response,
because arteries from pan-cytopenic rabbits had entirely normal
vasomotor function even after high doses of
2x1010 fp AV
vector.18 Thus, we believe
that the normal vasomotor profile of E1/E4-AV–treated vessels is
a reflection of the virus’ reduced toxicity and the corresponding
diminished vascular inflammation. The reduced intimal thickening in the
E1/E4-AV vector–treated vessels is likely a benefit of the
reduced inflammatory response. Endothelial injury
promotes endothelial activation, platelet
deposition, and inflammatory cell infiltration, leading to a release of
growth factors and chemokines that in turn stimulate vascular smooth
muscle growth and migration into the intima.
The reduced inflammatory response observed in the
E1/E4-AV–treated vessels is consistent with the
previously published studies examining the hepatic gene transfer after
intravenous delivery of the E1/E4-AV vector in
mice.32 However, in the
latter studies, the E1/E4-AV vector–treated mice demonstrated
significantly prolonged hepatic transgene expression (>7 months)
compared with the E1-AV vector (<2 months). In the present
studies, we observed only improved transgene expression with the
E1/E4-AV vector at 10 days after gene transfer. Essentially, no
transgene expression was observed at 28 days with either vector.
Similar results were obtained by 3 different methodologies:
lacZ-staining of the vessels, anti–ß-gal ELISA, and PCR detection of
the AV vectors. The drop-off in viral genomes, determined by using PCR,
indicates that the loss of ß-gal–staining resulted from the
clearance of the transduced cells. This may be attributable to the
observed residual inflammation, which although is substantially less
than the E1-AV vector, is nevertheless greater than that observed in
sham-treated vessels. The residual inflammation may be the result of
continued low-level viral antigen expression despite the multiple
lethal mutations. Although no L3 gene (hexon protein) expression and
greatly reduced L5 (fiber protein) gene expression is observed in
E1/E4-AV–transduced cells in vitro, this low level of late gene
expression may result in the expression of the immunogenic adenovirus
fiber protein. This may in turn be responsible for the observed loss of
adenoviral genomes from the E1/E4-AV–transduced arteries over
time. The gutless or helper-dependent adenoviral vectors that are
deleted of all genes encoding adenoviral
proteins26 33 34
would not experience this problem. It should be noted however that
detectable levels of E1-AV are frequently found to contaminate
preparations of the gutless vectors, and it is unclear how this would
impact vessel biology. An immune response to the xenoprotein ß-gal
may also be contributing to the observed residual inflammation.
Previous studies using recombinant adenoviruses expressing either
species-homologous genes35
or animals that are tolerant to the foreign
transgenes27 36
have suggested that the observed antiviral immune responses stem from
both viral- and transgene-specific responses. Furthermore, these
studies suggest that reducing either the viral or transgene component
can significantly improve the persistence of transgene expression. The
impact of the ß-gal transgene on vascular biology however is unclear
as our previous observations with a first-generation empty vector
suggests that it promoted similar levels of inflammation as an
identical vector containing the ß-gal
transgene.18
The demonstrated improvements in reduced inflammatory
responses, neointimal thickening, impairment of vessel
function, and improved persistence in transgene expression with the
E1/E4-AV vector are encouraging. The biology of this vector
suggests that the E1/E4-AV vector may be particularly well suited
in the treatment of vascular conditions requiring only transient gene
expression, for example, the genetic modification of vein grafts. We
hypothesize that antiproliferative and/or anti-inflammatory transgenes
should give substantially better results in vein graft models with the
E1/E4-AV vector than in an E1-AV vector. Further studies are
underway to examine this
possibility.
|
Acknowledgments |
|---|
This research was sponsored and supported by Cell Genesys Inc. The authors thank C. Hokanson for technical support.
|
Footnotes |
|---|
Original received June 29, 2000; resubmission received March 28, 2001; revised resubmission received April 3, 2001; accepted April 3, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ, Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell proliferation after arterial injury [see comments]. Science. 1994;265:781–784.
2. Claudio P, Fratta L, Farina F, Howard C, Stassi G, Numata S, Pacilio C, Davis A, Lavitrano M, Volpe M, Wilson J, Trimarco B, Giordano A, Condorelli G. Adenoviral RB2/p130 gene transfer inhibits smooth muscle cell proliferation and prevents restenosis after angioplasty. Circ Res. 1999;85:1032–1039.
3. Macejak D, Lin H, Webb S, Chase J, Jensen K, Jarvis T, Leiden J, Couture L. Adenovirus-mediated expression of a ribozyme to c-myb mRNA inhibits smooth muscle cell proliferation and neointima formation in vivo. J Virol. 1999;73:7745–7751.
4. Harrell RL, Rajanayagam S, Doanes AM, Guzman RJ, Hirschowitz EA, Crystal RG, Epstein SE, Finkel T. Inhibition of vascular smooth muscle cell proliferation and neointimal accumulation by adenovirus-mediated gene transfer of cytosine deaminase. Circulation. 1997;96:621–627.
5. Chang MW, Ohno T, Gordon D, Lu MM, Nabel GJ, Nabel EG, Leiden JM. Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery. Mol Med. 1995;1:172–181.
6. Chang M, Barr E, Lu M, Barton K, Leiden J. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96:2260–2268.
7. Chang M, Barr E, Seltzer J, Jiang Y, Nabel G, Nabel E, Parmacek M, Leiden J. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science. 1995;267:518–521.
8. Guzman RJ, Hirschowitz EA, Brody SL, Crystal RG, Epstein SE, Finkel T. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci U S A. 1994;91:10732–10736.
9. Waugh J, Kattash M, Li J, Yuksel E, Kuo M, Lussier M, Weinfeld A, Saxena R, Rabinovsky E, Thung S, Woo S, Shenaq S. Gene therapy to promote thromboresistance: local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model. Proc Natl Acad Sci U S A. 1999;96:1065–1070.
10. Mann MJ, Gibbons GH, Tsao PS, von der Leyen HE, Cooke JP, Buitrago R, Kernoff R, Dzau VJ. Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts. J Clin Invest. 1997;99:1295–1301.
11. Mann M, Whittemore A, Donaldson M, Belkin M, Conte M, Polak J, Orav E, Ehsan A, DellAcqua G, Dzau V. Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet. 1999;354:1493–1498.
12. Channon K, Fulton G, Gray J, Annex B, Shetty G, Blazing M, Peters K, Hagen P, George S. Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels. Cardiovasc Res. 1997;35:505–513.
13. Qian H, Neplioueva V, Shetty G, Channon K, George S. Nitric oxide synthase gene therapy rapidly reduces adhesion molecule expression and inflammatory cell infiltration in carotid arteries of cholesterol-fed rabbits. Circulation. 1999;99:2979–2982.
14. George S, Johnson J, Angelini G, Newby A, Baker A. Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein. Hum Gene Ther. 1998;9:867–877.
15. George S, Lloyd C, Angelini G, Newby A, Baker A. Inhibition of late vein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3. Circulation. 2000;101:296–304.
16. Bai H, Morishita R, Kida I, Yamakawa T, Zhang W, Aoki M, Matsushita H, Noda A, Nagai R, Kaneda Y, Higaki J, Ogihara T, Sawa Y, Matsuda H. Inhibition of intimal hyperplasia after vein grafting by in vivo transfer of human senescent cell-derived inhibitor-1 gene. Gene Ther. 1998;5:761–769.
17. Newman K, Dunn P, Owens J, Schulick A, Virmani R, Sukhova G, Libby P, Dichek D. 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.
18. Channon K, Qian H, Youngblood S, Olmez E, Shetty G, Neplioueva V, Blazing M, George S. 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.
19. Yang Y, Haecker S, Su Q, Wilson J. Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle. Hum Mol Genet. 1996;5:1703–1712.
20. Jooss K, Turka L, Wilson J. Blunting of immune responses to adenoviral vectors in mouse liver and lung with CTLA4Ig. Gene Ther. 1998;5:309–319.
21. Jooss K, Ertl H, Wilson J. Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovirus vectors delivered to mouse liver. J Virol. 1998;72:2945–2954.
22. Kay M, Meuse L, Gown A, Linsley P, Hollenbaugh D, Aruffo A, Ochs H, Wilson C. Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver. Proc Natl Acad Sci U S A. 1997;94:4686–4691.
23. Chirmule N, Raper S, Burkly L, Thomas D, Tazelaar J, Hughes J, Wilson J. Readministration of adenovirus vector in nonhuman primate lungs by blockade of CD40-CD40 ligand interactions. J Virol. 2000;74:3345–3352.
24. Chirmule N, Truneh A, Haecker S, Tazelaar J, Gao G, Raper S, Hughes J, Wilson J. Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody. J Immunol. 1999;163:448–455.
25. Krougliak V, Graham F. Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants. Hum Gene Ther. 1995;6:1575–1586.
26. Mitani K, Graham F, Caskey C, Kochanek S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci U S A. 1995;92:3854–3858.
27. Gao G, Yang Y, Wilson J. Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J Virol. 1996;70:8934–8943.
28. Engelhardt J, Ye X, Doranz B, Wilson J. 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.
29. Brough D, Lizonova A, Hsu C, Kulesa V, Kovesdi I. A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4. J Virol. 1996;70:6497–6501.
30. Wang Q, Jia X, Finer M. A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene-region deletions. Gene Ther. 1995;2:775–783.
31. Wang Q, Finer M. Second-generation adenovirus vectors. Nat Med. 1996;2:714–716.
32.
Wang Q, Greenburg
G, Bunch D, Farson D, Finer M. Persistent transgene expression in mouse
liver following in vivo gene transfer with a E1/E4 adenovirus
vector. Gene Ther. 1997;4:393–400.
33. Morsy M, Gu M, Motzel S, Zhao J, Lin Q, Su Q, Allen H, Ranlin L, Parks R, Graham F, Kochanek S, Bett A, Caskey C. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc Natl Acad Sci U S A. 1998;95:7866–7871.
34. Parks R, Chen L, Anton M, Sankar U, Rudnicki M, Graham F. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A. 1996;93:13565–13570.
35. Tripathy S, Black H, Goldwasser E, Leiden J. 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.
36. Dedieu J, Vigne E, Torrent C, Jullien C, Mahfouz I, Caillaud J, Aubailly N, Orsini C, Guillaume J, Opolon P, Delaere P, Perricaudet M, Yeh P. Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses. J Virol. 1997;71:4626–637.
This article has been cited by other articles:
 |
|
 |
|
  P. Roy-Chaudhury, V. P. Sukhatme, and A. K. Cheung Hemodialysis Vascular Access Dysfunction: A Cellular and Molecular Viewpoint J. Am. Soc. Nephrol., April 1, 2006; 17(4): 1112 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Murata, M. Hori, S. Lee, A. Nakamura, K. Kohama, H. Karaki, and H. Ozaki Vascular Endothelium Has a Local Anti-Adenovirus Vector System and Glucocorticoid Optimizes Its Gene Transduction Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1796 - 1803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. G. Melo, M. Gnecchi, A. S. Pachori, D. Kong, K. Wang, X. Liu, R. E. Pratt, and V. J. Dzau Endothelium-Targeted Gene and Cell-Based Therapies for Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1761 - 1774. [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]
|
|
|
|
 |
|
 F. Zhang, N. R. Hackett, G. Lam, J. Cheng, R. Pergolizzi, L. Luo, S. V. Shmelkov, J. Edelberg, R. G. Crystal, and S. Rafii Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells Blood, September 15, 2003; 102(6): 2115 - 2121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Li, L. M. Fan, A. Shah, and G. Brooks Targeting {alpha}v{beta}3 and {alpha}5{beta}1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-{beta}1 Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H1123 - H1131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. V. Tsui, A. Camrud, J. Mondesire, P. Carlson, N. Zayek, L. Camrud, B. Donahue, S. Bauer, A. Lin, D. Frey, et al. p27-p16 Fusion Gene Inhibits Angioplasty-Induced Neointimal Hyperplasia and Coronary Artery Occlusion Circ. Res., August 17, 2001; 89(4): 323 - 328. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||