![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Virology, July 1999, p. 6141-6146, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Frequency and Stability of Chromosomal Integration
of Adenovirus Vectors
Airi
Harui,1
Shinobu
Suzuki,1
Stefan
Kochanek,2 and
Kohnosuke
Mitani1,3,*
Department of Microbiology, Immunology and
Molecular Genetics, UCLA School of Medicine,1
and Jonsson Comprehensive Cancer
Center,3 Los Angeles, California 90095-1747, and
Center for Molecular Medicine (ZMMK), University of
Cologne, 50931 Cologne, Germany2
Received 20 November 1998/Accepted 22 March 1999
 |
ABSTRACT |
One of the limitations of adenovirus vectors is the lack of
machinery necessary for their integration into host chromosomes, resulting in short-term gene expression in dividing cells. We analyzed
frequencies of integration and persistence of gene expression from
integrated adenovirus vectors. Both E1-substituted and helper-dependent adenovirus vectors achieved similar integration efficiencies of ~10
3 to 105 per cell, with the
helper-dependent vector showing slightly higher efficiencies. In stable
cell pools, gene expression of the integrated vector persisted for at
least 50 cell divisions without selection. However, some stable cell
clones showed changes in gene expression, which were accompanied by
structural changes in the integrated vector DNA.
| |
TEXT |
Recombinant adenovirus vectors are
attracting increasing attention as in vivo gene transfer vehicles for
human gene therapy (2, 31, 36). However, one of the
limitations of E1-substituted adenovirus vectors currently used in most
clinical gene therapy protocols is the relatively short-term expression
of the transferred gene in vivo. E1-substituted vectors express viral
antigens that induce a cytotoxic-T-lymphocyte-mediated immune response
against the vector-transduced cells by the host, resulting in
inflammation and short-term gene expression from the vector in vivo
(3, 28, 32-35). To overcome this problem, we and others
have recently developed a helper-dependent (gutless) adenovirus vector
by removing all the viral coding sequences from adenovirus vector DNA
(7, 11, 12, 15, 19). Such vectors are therefore expected to minimize the immune responses of the host that would cause rejection of
the transduced cells (20, 24). However, even with this system, vector DNA is eventually lost in dividing cells because although adenoviruses exist as multiple episomal copies in the infected
cell nuclei, they lack the machinery necessary for integration into
host chromosomes. Furthermore, activation of T-helper cells and B cells
in response to viral capsid proteins produces neutralizing antibodies
that block the efficient readministration of vector (13,
37). Therefore, further improvement of an adenovirus vector that
replicates or efficiently integrates into host chromosomes is required
to obtain long-term expression, even in dividing cells. Although it is
known that a wild-type adenovirus rarely integrates into the
chromosomes of cells that are not permissive for viral DNA replication,
there have not yet been any extensive investigations of how frequently
replication-incompetent adenovirus vectors integrate into host
chromosomes. In this study, we analyzed frequencies of integration of
E1-substituted and helper-dependent adenovirus vectors and stability of
gene expression from the integrated vectors.
Integration efficiencies of E1-substituted and helper-dependent
adenovirus vectors in cell lines.
To compare the efficiencies of
integration of the E1-substituted and helper-dependent adenovirus
vectors, we rescued both types of vectors with the 
-geo
marker gene, a fusion of the E. coli -galactosidase
(-Gal) gene and the neomycin phosphotransferase II gene
(neo) (9) that is driven by the SR
promoter
(27) (SR-geo) (Fig.
1). The SR-geo marker
gene cassette (19) was subcloned into an adenovirus transfer
plasmid, pXCX2 (26). AdSR-geo, an E1-substituted
adenovirus vector (
E1 vector), was rescued by cotransfection into
the 293 cell line (Microbix, Toronto, Ontario, Canada) of the plasmid
with pJM17 (18). The virus was then plaque isolated,
propagated, and purified as described previously (10). The
titer of the vector was 1.2 × 109 PFU/ml on 293 cells. A helper-dependent adenovirus vector DNA was constructed as
follows. First, an AvrII-SmaI fragment of pFG140 (18) encompassing the junction of the ligated right end (452 bp) and left end (1,009 bp) of Ad5 was subcloned into an
XbaI site of the charomid 9-22 vector (23). This
adenovirus sequence contains two inverted terminal repeats and the
packaging signal but does not encode any intact open reading frames of
the parental human adenovirus type 5 (Ad5). Second, the
SR-geo cassette was subcloned into a SmaI
site of the plasmid. The helper-dependent Ad
SR-geo vector
(Ad vector) was rescued by cotransfection into 293 cells of the
resultant plasmid with a helper-virus DNA-terminal protein complex.
AdHprt, an adenovirus vector with E1 and E3 deleted and with a
nonfunctional genomic sequence from the mouse Hprt locus,
was used as a helper virus. The vector was propagated and purified as
described previously (12, 19). After three rounds of
purification by a CsCl density gradient, the titer of the vector was
measured in situ, using
5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) as a
substrate, as previously described (17). The titer of the
vector on the African green monkey cell line COS7 (American Type
Culture Collection [ATCC], Rockville, Md.) was 2.4 × 109 -Gal-transducing units (BTU)/ml, and that of the
helper on 293 cells, as measured by a plaque assay, was 2.2 × 107 PFU/ml. Therefore, the vector stock contained 0.9%
helper virus contamination.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 1.
Structure of adenovirus vectors. H,
HindIII sites used for Southern hybridization analysis
of the structure of integrated vectors.
|
|
The human cell lines HeLa (ATCC), HT1080 (ATCC), and KB (ATCC), the
African green monkey kidney cell lines CV-1 (ATCC) and Vero (provided
by Harumi Kasamatsu), the baby hamster kidney (BHK) cell line (provided
by Debi Nayak), the Chinese hamster ovary (CHO) cell line (provided by
Owen N. Witte), and the mouse cell line NIH 3T3 (ATCC) were infected
with the vectors at 10 PFU/cell for the E1 vector or 10 BTU/cell for
the Ad vector. At 48 h postinfection, the infected cells were
diluted and replated onto 96-well plates at densities of
105, 104, and 103 cells/plate. To
avoid low plating efficiencies at low cell densities, the total cell
numbers in each plate were adjusted to 105 by adding
uninfected cells. After 24 h, medium containing G418 was added.
The final concentration of G418 was 500 µg/ml (HeLa, HT1080, BHK, and
NIH 3T3 cells) or 1,000 µg/ml (KB, CHO, CV-1, and Vero cells).
G418-containing medium was added again 1 week later, and the
G418-resistant colonies were counted and integration efficiencies were
calculated 4 weeks postinfection. The results are summarized in Table
1. In most of the cell lines, both
vectors integrated efficiently at frequencies of 104 to
105, but we occasionally observed a higher efficiency of
103 in HT1080 and KB cells. CHO cells showed relatively
high integration efficiencies, particularly with the Ad vector,
which achieved levels above 1%. In the experiments with HeLa, KB, and
CHO cell lines, in which both types of vectors were tested in parallel, integration frequencies were severalfold higher with the Ad vector than with the E1 vector, but both vectors showed similar
efficiencies in HT1080 cells.
The relation of the PFU titer of the E1 vector to the BTU titer of
the Ad vector was determined as follows. Infected HeLa cells were
harvested 4 h postinfection, at which time viral DNA synthesis had
not yet started, and total DNA was extracted. The DNA was digested with
HindIII and subjected to Southern hybridization with a
-Gal fragment (nucleotides 118 to 581 of ECLACZ; GenBank no. V00296)
as a probe. Based on the intensity of the signal measured by a Storm
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.), the copy number
of the Ad vector at 10 BTU/cell is not as high as that of the E1
vector at 10 PFU/cell but corresponds to the copy number at 6.4 PFU/cell. Therefore, the integration efficiency of the Ad vector in
Table 1 might be an underestimate, compared to that of the E1 vector.
Integration efficiencies of the adenovirus vector that were assessed by
G418 resistance could be affected by vector expression levels of the
selectable marker gene, -geo (25), and/or by the ability of vector DNA to integrate into chromosomes. To distinguish between these two possibilities, the infected HeLa, HT1080, KB, and CHO
cells were plated into 100-mm dishes and subjected to G418 selection to
obtain pools of stably transduced cells. Cell extracts were prepared
from these cell pools, and the expression levels of the
-geo marker gene were compared. The levels of -Gal activity showed no correlation to vector or cell types (data not shown), indicating that there are no significant differences in the
levels of expression of SR-geo once it integrates into
a host chromosome, regardless of the vector backbones used. These results suggest that more efficient integration by the Ad vector is
mainly due to the stronger ability of the Ad vector to integrate into cellular chromosomes. The higher integration efficiencies of the
Ad vector can be attributed to the nature of completely nonviral
genomic sequences or to the lack of leaky expression of viral genes,
some of which inhibit normal cellular machinery, unlike those in the
E1 vector.
Although wild-type adenovirus does not integrate into the chromosomes
of the permissive cells because its infection leads to lytic infection,
it can integrate in nonpermissive cells (e.g., hamster cells infected
with Ad12) (for a review, see reference 6). In
addition, adenovirus mutants that code for a temperature-sensitive DNA-binding protein yield stable cell clones at nonpermissive temperatures (8). Chromosomal integration of an
E1-substituted adenovirus vector in rat and simian cells has previously
been reported (30). At a multiplicity of infection (MOI) of
200, the integration efficiencies were 0.4% for Rat2 cells and 0.75% for CV-1 cells. More recently, stable integration of E1-substituted adenovirus vector with the -gal marker gene in mouse NIH
3T3, human A549, and primary human cells has been reported
(38). Although the integration efficiency was 15% in these
cell lines when ionizing radiation was used, the efficiency without
radiation was not reported.
In our study, the E1 and Ad vectors were used to investigate the
frequencies of integration of adenovirus vectors in different cell
types (Table 1). Human epidermoid cell lines such as HeLa and KB are
known to be permissive for viral DNA replication of wild-type Ad5. BHK
and Vero cells are semipermissive, and CV-1 and CHO cells are
nonpermissive (16). Most of the cell lines infected by the
E1 vector showed similar integration efficiencies of
~103 to 105 (Table 1), suggesting that
factors determining cellular permissiveness do not affect the
integration of adenovirus vectors. Under these conditions (MOI of 10),
only up to 10 viral DNA molecules entered per cell, in contrast to
approximately 106 DNA molecules per cell transferred by the
calcium phosphate transfection method, in which 2.2 to 6.4% of DNA is
internalized into the nucleus (21). Our results indicate
that the ability of adenovirus DNA to integrate into host chromosomes
seems extremely high compared to that of naked plasmid DNA, even though
the end is protected by the terminal protein.
Sustained gene expression from integrated adenovirus vector.
To analyze the persistence of gene expression from integrated
adenovirus vectors, the pools and clones of stably transduced cells,
obtained as described above, were cultured without G418 selection.
Expression of -Gal in these cells was detected by X-Gal staining at
every five passages (Fig. 2). In most of
the stable cell pools, -Gal expression did not diminish over at
least 15 passages, which corresponds to approximately 50 cell divisions (Fig. 2A and B). There were no significant differences in the time-course profiles of marker gene expression between the E1 and
Ad vectors. For more quantitative analysis, the levels of -Gal
enzymatic activity were also measured in triplicate (with the
luminescent -Gal gene reporter system 2; Clontech, Palo Alto, Calif.) before and after the series of passages without G418 (Table 2). The amount of protein in each sample
was standardized by the Bradford method (Bio-Rad protein assay;
Bio-Rad, Hercules, Calif.). Each cell pool showed different patterns of
stability of -Gal expression from the integrated vectors. In HT1080
cells, the levels of -Gal activity tended to decrease with both the E1 (0.5- and 0.7-fold) and Ad (0.4- and 0.6-fold) vectors. In KB
cells, the levels of -Gal activity remained the same for both the
E1 (1.0- and 1.2-fold) and Ad (0.9- and 1.4-fold) vectors. In CHO
cells, the levels of -Gal activity from the integrated E1 vector
(0.7- and 0.5-fold), but not from the Ad vector (1.1- and 0.7-fold),
tended to decrease. We also analyzed the persistence of gene expression
of individual stable HeLa clones (Fig. 2C and D). In two of five HeLa
cell clones with stably integrated E1 vectors, the level of -Gal
activity decreased from high to low over time (clones 2 and 4) (Fig.
2C). Interestingly, in HeLa clone 5, which was transduced by the Ad
vector, the percentage of -Gal-positive cells increased dramatically
over time (Fig. 2D). This observation is in contrast to the expression
of viral genes from integrated wild-type adenovirus, which is often
shut off by methylation (4-6).
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 2.
-Gal expression from the integrated vectors over
passages of the cells infected with E1 vector (A and C) and Ad
vector (B and D) without G418 selection. The stable cell pools (A and
B) and HeLa clones (C and D) were maintained without G418 selection and
stained by X-Gal every five passages; the percentages of X-Gal-positive
cells were determined by microscopic observation. Two independent pools
were analyzed for each cell line.
|
|
Structure of the integrated vector.
The structure of
adenovirus DNA integrated into host chromosomes is known to vary,
depending on the cell line and the type of virus. Rat embryonic cells
transformed by Ad5 usually contain only the left end of the genome,
whereas cell lines transformed by temperature-sensitive adenovirus
mutants at semi- or nonpermissive temperatures often contain multiple
copies of all or most of the adenovirus genome (6). To
analyze the structure of integrated adenovirus vectors, genomic DNA was
extracted from stably transduced HeLa cell clones, digested with
HindIII, and subjected to Southern hybridization.
Fragments of the neo gene, the right end of Ad5 (nucleotides
35032 to 35780 of ADRCOMPGEN; GenBank no. M73260 and M29978), and
full-length Ad5 DNA were used as probes (Fig. 1). The right-end probe
hybridized with the DNA fragment between the rightmost
HindIII site in the adenovirus genome and a
HindIII site to the right of the integration site, which
produced a band representing a size unique to the integration site. In
E1 stable clones, the right-end probe hybridized with one band in
three of five clones, indicating a single-copy integration of the right end of the vector. However, clones 2 and 5 had three and two bands, respectively, indicating multiple integrations of the right end in
these clones (Fig. 3A). Similarly, the
neo probe with E1 integrants produced a band representing
a size unique to a left-end integration junction. Contrary to the
results with the right-end probe, clones 1, 2, and 5 showed single-copy
integration, while clones 3 and 4 showed two integrants each (Fig. 3C).
Clone 1 had one additional faint band hybridizing with the right-end
probe. This faint band might indicate that in some cells the integrated
vector is rearranged. Finally, the full-length adenovirus probe should
produce eight bands specific for the full-length vector, with two
additional bands corresponding to the junctions of integration sites at
both ends of the vector (Fig. 1). All of the clones showed the bands common to AdSR-geo viral DNA (Fig. 3E, lanes 1, 2, 5, and 6). These results indicate that all of the HeLa E1 stable clones had at least one copy of full-length vector integrated into their chromosomes, which was often accompanied by additional integration events. In the case of the stable Ad clones, all the integrants showed a single band with both the right-end and the neo
probes (Figs. 3B and D), suggesting that most of the stable cell clones had a single-copy integration. Clone 2 had two additional faint bands
hybridizing with the right-end probe, suggesting rearrangement of
integrated vectors in some cells. The neo probe should
produce a 26-kb internal fragment encompassing the
SR-geo cassette and the stuffer sequence consisting of
repeats of a 2-kb fragment from pBR (Fig. 1). However, at least two
clones (clones 3 and 5) (Fig. 3D) showed fragments smaller than those
of other clones. Therefore, in these clones, an internal fragment was
deleted before or after integration. The deletions might be associated
with the nature of the repetitive sequences of the fragment. In CV-1
cell clones, E1 vector showed patterns of integration similar to
those in HeLa cells (i.e., a single full-length copy with an additional end sequence). On the other hand, Ad vector tended to integrate at
multiple sites in many clones but, unlike in HeLa cells, no internal
deletion was detected by Southern analysis (data not shown). It is not
clear why the E1 and Ad vectors show distinct patterns of
integration.
View larger version (35K):
[in this window]
[in a new window]
|
FIG. 3.
Southern analysis of integrated vector DNA. DNA from
HeLa cell clones infected with the E1 (A, C, and E) or Ad (B, D,
and E) vector was digested with HindIII and subjected to
Southern hybridization with the right-end (A and B), neo (C
and D), and full-length Ad5 DNA (E) probes. (A and C) Lanes 1 to 3, E1 clones 1 to 3, respectively; lane 4, clone 4 before the passages
without selection; lane 5, clone 4 after the passages; lane 6, clone 5 before the passages; lane 7, clone 5 after the passages; lane 8, AdSR-geo DNA. (B and D) Lanes 1 to 4, Ad clones 1 to 4, respectively; lane 5, clone 5 before the passages without selection;
lane 6, clone 5 after the passages. (E) Lane 1, E1 clone 5; lane 2, E1 clone 3; lane 3, Ad clone 3; lane 4, Ad clone 4, lane 5, E1 clone 1; lane 6, E1 clone 2; lane 7, uninfected HeLa; lane 8, AdSR-geo DNA.
|
|
We also examined the changes in the structure of integrated vector in
three HeLa clones before and after passages without selection. In E1
clone 4, the integrated vector signal disappeared, as indicated by both
probes after 15 passages, consistent with the decrease in levels of
-Gal-positive cells from 95 to 0% (Fig. 3A and C, lanes 4 and 5).
In E1 clone 5, which had a moderate decrease in -Gal-positive
cells (from 90 to 70%), one of the two fragments hybridizing with the
right-end probe disappeared (Fig. 3A, lanes 6 and 7), while the only
band that hybridized with the neo probe remained unaltered
before and after passages without selection (Fig. 3C, lanes 6 and 7).
On the other hand, in the Ad stable clone 5, which had increased
activity (from 1 to 100%), the integrated vector showed a rearranged
pattern after 15 passages (Fig. 3B and D, lanes 5 and 6). This
rearrangement might account for the increased -Gal activity.
Rearrangement of integrated viral DNA was also documented in hamster
cells with stably integrated Ad12 (14).
In summary, although adenovirus vectors integrate into host chromosomes
relatively efficiently, unlike retroviral integration, most of the
stable clones have an extra fragment(s) of the vector or deleted
vector. Gene expression from the integrated vector is relatively
stable. However, integrated vectors sometimes become further
rearranged, resulting in an altered level of gene expression. Adenovirus vectors can infect a variety of cell types at very high
efficiencies. Our results suggest that it might be possible to use an
adenovirus vector to establish stable cell clones in cells which are
refractory to other gene delivery methods. Considering the somewhat
unstable nature of integrated adenovirus vector DNA, selection
strategies for the integrated DNA might be needed to obtain long-term
expression. It is known that gene expression from retroviral vectors is
shut off in some primary cell cultures. Therefore, it would be of
interest to analyze the integration frequencies of adenovirus vectors
and the longevity of gene expression, especially in primary cultures
such as cultures of hematopoietic cells, which are one of the main
targets for human gene therapy. On the other hand, because one of the
advantages of an adenovirus vector for human gene therapy is its rare
chromosomal integration, thereby circumventing potential insertional
mutagenesis of cancer-related genes, it would be important to determine
how frequently an adenovirus vector integrates in vivo. There are
reports suggesting that stable integration of adenovirus vectors into
the host chromosome occurs after in vivo gene transfer in animals
(1, 22). Efficient production in transgenic mice by
adenovirus gene transfer into fertilized eggs was also reported
(29). Therefore, considering the high MOIs usually used for
in vivo gene transfer, elucidation of adenovirus integration in vivo is
a very important issue for evaluating the safety of adenovirus vectors
for human gene therapy.
| |
ACKNOWLEDGMENTS |
A.H. and S.S. contributed equally to this work.
We thank Harumi Kasamatsu, Debi Nayak, and Owen N. Witte for providing
cell lines, Frank L. Graham for providing the pXCX2 and pJM17 plasmids,
Izumu Saito for providing the charomid vector, Arnie Berk for critical
discussion and for reading the manuscript, Wendy Aft for preparation of
the manuscript, Amit Oberai for generating preliminary data, and Lena
Kim for technical support.
This work was supported by NIH grant AI-42214.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Immunology and Molecular Genetics, UCLA School of
Medicine, Box 951747, Los Angeles, CA 90095-1747. Phone: (310)
267-2031. Fax: (310) 206-3865. E-mail: mitani{at}ucla.edu.
| |
REFERENCES |
| 1.
|
Brown, G. R.,
D. L. Thiele,
M. Silva, and B. Beutler.
1997.
Adenoviral vectors given intravenously to immunocompromised mice yield stable transduction of the colonic epithelium.
Gastroenterology
112:1586-1594[Medline].
|
| 2.
|
Crystal, R. G.
1995.
Transfer of genes to humans: early lessons and obstacles to success.
Science
270:404-410[Abstract/Free Full Text].
|
| 3.
|
Dai, Y.,
E. M. Schwarz,
D. Gu,
W. W. Zhang,
N. Sarvetnick, and I. M. Verma.
1995.
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. USA
92:1401-1405[Abstract/Free Full Text].
|
| 4.
|
Doerfler, W.
1991.
Abortive infection and malignant transformation by adenoviruses: integration of viral DNA and control of viral gene expression by specific patterns of DNA methylation.
Adv. Virus Res.
39:89-128[Medline].
|
| 5.
|
Doerfler, W.
1984.
DNA methylation and its functional significance: studies on the adenovirus system.
Curr. Top. Microbiol. Immunol.
108:79-98[Medline].
|
| 6.
|
Doerfler, W.
1982.
Uptake, fixation, and expression of foreign DNA in mammalian cells: the organization of integrated adenovirus DNA sequences.
Curr. Top. Microbiol. Immunol.
101:127-194[Medline].
|
| 7.
|
Fisher, K. J.,
H. Choi,
J. Burda,
S. Chen, and J. M. Wilson.
1996.
Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis.
Virology
217:11-22[Medline].
|
| 8.
|
Fisher, P. B.,
L. E. Babiss,
I. B. Weinstein, and H. S. Ginsberg.
1982.
Analysis of type 5 adenovirus transformation with a cloned rat embryo cell line (CREF).
Proc. Natl. Acad. Sci. USA
79:3527-3531[Abstract/Free Full Text].
|
| 9.
|
Friedrich, G., and P. Soriano.
1991.
Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.
Genes Dev.
5:1513-1523[Abstract/Free Full Text].
|
| 10.
|
Graham, F. L., and L. Prevec.
1995.
Methods for construction of adenovirus vectors.
Mol. Biotechnol.
3:207-220[Medline].
|
| 11.
|
Haecker, S. E.,
H. H. Stedman,
R. J. Balice-Gordon,
D. B. Smith,
J. P. Greelish,
M. A. Mitchell,
A. Wells,
H. L. Sweeney, and J. M. Wilson.
1996.
In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes.
Hum. Gene Ther.
7:1907-1914[Medline].
|
| 12.
|
Kochanek, S.,
P. R. Clemens,
K. Mitani,
H.-H. Chen,
S. Chan, and C. T. Caskey.
1996.
A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and -galactosidase.
Proc. Natl. Acad. Sci. USA
93:5731-5736[Abstract/Free Full Text].
|
| 13.
|
Kozarsky, K. F.,
D. R. McKinley,
L. L. Austin,
S. E. Raper,
L. D. Stratford-Perricaudet, and J. M. Wilson.
1994.
In vivo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses.
J. Biol. Chem.
269:13695-13702[Abstract/Free Full Text].
|
| 14.
|
Kuhlmann, I., and W. Doerfler.
1983.
Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA.
J. Virol.
47:631-636[Abstract/Free Full Text].
|
| 15.
|
Kumar-Singh, R., and J. S. Chamberlain.
1996.
Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells.
Hum. Mol. Genet.
5:913-921[Abstract/Free Full Text].
|
| 16.
|
Lucher, L. A.
1995.
Abortive adenovirus infection and host range determinants.
Curr. Top. Microbiol. Immunol.
199:119-152.
|
| 17.
|
MacGregor, G. R.,
A. E. Mogg,
J. F. Burke, and C. T. Caskey.
1987.
Histochemical staining of clonal mammalian cell lines expressing E. coli -galactosidase indicates heterogeneous expression of the bacterial gene.
Somatic Cell Mol. Genet.
13:253-265[Medline].
|
| 18.
|
McGrory, W. J.,
D. S. Bautista, and F. J. Graham.
1988.
A simple technique for the rescue of early region I mutants into infectious human adenovirus type 5.
Virology
163:614-617[Medline].
|
| 19.
|
Mitani, K.,
F. L. Graham,
C. T. Caskey, and S. Kochanek.
1995.
Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.
Proc. Natl. Acad. Sci. USA
92:3854-3858[Abstract/Free Full Text].
|
| 20.
|
Morsy, M. A.,
M. Gu,
S. Motzel,
J. Zhao,
J. Lin,
Q. Su,
H. Allen,
L. Franlin,
R. J. Parks,
F. L. Graham,
S. Kochanek,
A. J. Bett, and C. T. Caskey.
1998.
An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene.
Proc. Natl. Acad. Sci. USA
95:7866-7871[Abstract/Free Full Text].
|
| 21.
|
Orrantia, E., and P. L. Chang.
1990.
Intracellular distribution of DNA internalized through calcium phosphate precipitation.
Exp. Cell Res.
190:170-174[Medline].
|
| 22.
|
Overturf, K.,
M. Al-Dhalimy,
C. N. Ou,
M. Finegold,
R. Tanguay,
A. Lieber,
M. Kay, and M. Grompe.
1997.
Adenovirus-mediated gene therapy in a mouse model of hereditary tyrosinemia type I.
Hum. Gene Ther.
8:513-521[Medline].
|
| 23.
|
Saito, I., and G. R. Stark.
1986.
Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments.
Proc. Natl. Acad. Sci. USA
83:8664-8668[Abstract/Free Full Text].
|
| 24.
|
Schiedner, G.,
N. Morral,
R. J. Parks,
Y. Wu,
S. C. Koopmans,
C. Langston,
F. L. Graham,
A. L. Beaudet, and S. Kochanek.
1998.
Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity.
Nat. Genet.
18:180-183[Medline].
|
| 25.
|
Soriano, P.,
C. Montgomery,
R. Geske, and A. Bradley.
1991.
Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice.
Cell
64:693-702[Medline].
|
| 26.
|
Spessot, R.,
K. Inchley,
T. M. Hupel, and S. Bacchetti.
1989.
Cloning of the herpes simplex virus ICP4 gene in an adenovirus vector: effects on adenovirus gene expression and replication.
Virology
168:378-387[Medline].
|
| 27.
|
Takebe, Y.,
M. Seiki,
J.-I. Fujisawa,
P. Hoy,
K. Yokota,
K.-I. Arai,
M. Yoshida, and N. Arai.
1988.
SR promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat.
Mol. Cell. Biol.
8:466-472[Abstract/Free Full Text].
|
| 28.
|
Tripathy, S. K.,
H. B. Black,
E. Goldwasser, and J. M. Leiden.
1996.
Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors.
Nat. Med.
2:545-550[Medline].
|
| 29.
|
Tsukui, T.,
Y. Kanegae,
I. Saito, and Y. Toyoda.
1996.
Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs.
Nat. Biotechnol.
14:982-985[Medline].
|
| 30.
|
Van Doren, K.,
D. Hanahan, and Y. Gluzman.
1984.
Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA.
J. Virol.
50:606-614[Abstract/Free Full Text].
|
| 31.
|
Verma, I. M., and N. Somia.
1997.
Gene therapy promises, problems and prospects.
Nature
389:239-242[Medline].
|
| 32.
|
Yang, Y.,
S. E. Haecker,
Q. Su, and J. M. Wilson.
1996.
Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle.
Hum. Mol. Genet.
5:1703-1712[Abstract/Free Full Text].
|
| 33.
|
Yang, Y.,
F. A. Nunes,
K. Berencsi,
E. E. Furth,
E. Gonczol, and J. M. Wilson.
1994.
Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.
Proc. Natl. Acad. Sci. USA
91:4407-4411[Abstract/Free Full Text].
|
| 34.
|
Yang, Y., and J. M. Wilson.
1995.
Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo.
J. Immunol.
155:2564-2570[Abstract].
|
| 35.
|
Yang, Y.,
Z. Xiang,
H. C. Ertl, and J. M. Wilson.
1995.
Upregulation of class I major histocompatibility complex antigens by interferon- is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.
Proc. Natl. Acad. Sci. USA
92:7257-7261[Abstract/Free Full Text].
|
| 36.
|
Yeh, P., and M. Perricaudet.
1997.
Advances in adenoviral vectors: from genetic engineering to their biology.
FASEB J.
11:615-623[Abstract].
|
| 37.
|
Yei, S.,
N. Mittereder,
K. Tang,
C. O. Sullivan, and B. C. Trapnell.
1994.
Adenovirus-mediated gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung.
Gene Ther.
1:192-200[Medline].
|
| 38.
|
Zeng, M.,
G. J. Cerniglia,
S. L. Eck, and C. W. Stevens.
1997.
High-efficiency stable gene transfer of adenovirus into mammalian cells using ionizing radiation.
Hum. Gene Ther.
8:1025-1032[Medline].
|
Journal of Virology, July 1999, p. 6141-6146, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Mali, P., Ye, Z., Hommond, H. H., Yu, X., Lin, J., Chen, G., Zou, J., Cheng, L.
(2008). Improved Efficiency and Pace of Generating Induced Pluripotent Stem Cells from Human Adult and Fetal Fibroblasts. Stem Cells
26: 1998-2005
[Abstract]
[Full Text]
-
Takehashi, M., Kanatsu-Shinohara, M., Inoue, K., Ogonuki, N., Miki, H., Toyokuni, S., Ogura, A., Shinohara, T.
(2007). Adenovirus-mediated gene delivery into mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA
104: 2596-2601
[Abstract]
[Full Text]
-
Schnepp, B. C., Jensen, R. L., Chen, C.-L., Johnson, P. R., Clark, K. R.
(2005). Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues. J. Virol.
79: 14793-14803
[Abstract]
[Full Text]
-
Ohbayashi, F., Balamotis, M. A., Kishimoto, A., Aizawa, E., Diaz, A., Hasty, P., Graham, F. L., Caskey, C. T., Mitani, K.
(2005). Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors. Proc. Natl. Acad. Sci. USA
102: 13628-13633
[Abstract]
[Full Text]
-
Wang, H., Shayakhmetov, D. M., Leege, T., Harkey, M., Li, Q., Papayannopoulou, T., Stamatoyannopolous, G., Lieber, A.
(2005). A Capsid-Modified Helper-Dependent Adenovirus Vector Containing the {beta}-Globin Locus Control Region Displays a Nonrandom Integration Pattern and Allows Stable, Erythroid-Specific Gene Expression. J. Virol.
79: 10999-11013
[Abstract]
[Full Text]
-
Duigou, G. J., Young, C. S. H.
(2005). Replication-Competent Adenovirus Formation in 293 Cells: the Recombination-Based Rate Is Influenced by Structure and Location of the Transgene Cassette and Not Increased by Overproduction of HsRad51, Rad51-Interacting, or E2F Family Proteins. J. Virol.
79: 5437-5444
[Abstract]
[Full Text]
-
Schakman, O., Gilson, H., de Coninck, V., Lause, P., Verniers, J., Havaux, X., Ketelslegers, J. M., Thissen, J. P.
(2005). Insulin-Like Growth Factor-I Gene Transfer by Electroporation Prevents Skeletal Muscle Atrophy in Glucocorticoid-Treated Rats. Endocrinology
146: 1789-1797
[Abstract]
[Full Text]
-
Dorigo, O., Gil, J. S., Gallaher, S. D., Tan, B. T., Castro, M. G., Lowenstein, P. R., Calos, M. P., Berk, A. J.
(2004). Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells. J. Virol.
78: 6556-6566
[Abstract]
[Full Text]
-
Hashimoto, M., Mikoshiba, K.
(2004). Neuronal Birthdate-Specific Gene Transfer with Adenoviral Vectors. J. Neurosci.
24: 286-296
[Abstract]
[Full Text]
-
Ehrhardt, A., Xu, H., Kay, M. A.
(2003). Episomal Persistence of Recombinant Adenoviral Vector Genomes during the Cell Cycle In Vivo. J. Virol.
77: 7689-7695
[Abstract]
[Full Text]
-
Schnepp, B. C., Clark, K. R., Klemanski, D. L., Pacak, C. A., Johnson, P. R.
(2003). Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle. J. Virol.
77: 3495-3504
[Abstract]
[Full Text]
-
Kubo, S., Mitani, K.
(2003). A New Hybrid System Capable of Efficient Lentiviral Vector Production and Stable Gene Transfer Mediated by a Single Helper-Dependent Adenoviral Vector. J. Virol.
77: 2964-2971
[Abstract]
[Full Text]
-
Roth, M. D., Cheng, Q., Harui, A., Basak, S. K., Mitani, K., Low, T. A., Kiertscher, S. M.
(2002). Helper-Dependent Adenoviral Vectors Efficiently Express Transgenes in Human Dendritic Cells but Still Stimulate Antiviral Immune Responses. J. Immunol.
169: 4651-4656
[Abstract]
[Full Text]
-
Gordon, J. W.
(2002). High Toxicity, Low Receptor Density, and Low Integration Frequency Severely Impede the Use of Adenovirus Vectors for Production of Transgenic Mice. Biol. Reprod.
67: 1172-1179
[Abstract]
[Full Text]
-
Gordon, J. W.
(2002). Adenovirus gene transfer vector toxicity to mouse embryos: implications for human IVF. Hum Reprod
17: 2380-2387
[Abstract]
[Full Text]
-
Hillgenberg, M., Tonnies, H., Strauss, M.
(2001). Chromosomal Integration Pattern of a Helper-Dependent Minimal Adenovirus Vector with a Selectable Marker Inserted into a 27.4-Kilobase Genomic Stuffer. J. Virol.
75: 9896-9908
[Abstract]
[Full Text]
-
Oka, K., Pastore, L., Kim, I.-H., Merched, A., Nomura, S., Lee, H.-J., Merched-Sauvage, M., Arden-Riley, C., Lee, B., Finegold, M., Beaudet, A., Chan, L.
(2001). Long-Term Stable Correction of Low-Density Lipoprotein Receptor-Deficient Mice With a Helper-Dependent Adenoviral Vector Expressing the Very Low-Density Lipoprotein Receptor. Circulation
103: 1274-1281
[Abstract]
[Full Text]
Top
Abstract
Text
