![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Virology, August 2001, p. 6969-6976, Vol. 75, No. 15
Departments of Pediatrics and Genetics,
Stanford University School of Medicine, Stanford, California 94305
Received 15 February 2001/Accepted 29 April 2001
Recombinant adeno-associated virus (rAAV) vectors stably transduce
hepatocytes in experimental animals. Although the vector genomes are
found both as extrachromosomes and as chromosomally integrated forms in
hepatocytes, the relative proportion of each has not yet been clearly
established. Using an in vivo assay based on the induction of
hepatocellular regeneration via a surgical two-thirds partial
hepatectomy, we have determined the proportion of integrated and
extrachromosomal rAAV genomes in mouse livers and their relative
contribution to stable gene expression in vivo. Plasma human
coagulation factor IX (hF.IX) levels in mice originating from a
chromosomally integrated hF.IX-expressing transposon vector remained
unchanged with hepatectomy. This was in sharp contrast to what was
observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of
hF.IX-expressing rAAV vectors. At doses of 2.4 × 1011
to 3.0 × 1011 vector genomes per mouse
(n = 12), hF.IX levels and the average number of
stably transduced vector genomes per cell decreased by 92 and 86%,
respectively, after hepatectomy. In a separate study, one
of three mice injected with a higher dose of rAAV had a higher
proportion (67%) of integrated genomes, the significance of which is
not known. Nevertheless, in general, these results indicate that, in
most cases, no more than ~10% of stably transduced genomes
integrated into host chromosomes in vivo. Additionally, the results
demonstrate that extrachromosomal, not integrated, genomes are the
major form of rAAV in the liver and are the primary source of
rAAV-mediated gene expression. This small fraction of integrated
genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but
also raises uncertainties as to whether rAAV-mediated hepatic gene
expression can persist lifelong after a single vector administration.
Adeno-associated virus (AAV) is a
replication-defective human parvovirus with a single-stranded (ss) DNA
genome of approximately 4.7 kb. Recombinant AAV (rAAV) vectors based on
AAV type 2 are of great interest in the field of gene therapy for
monogenic diseases because they can safely direct persistent transgene
expression from transduced tissues in both experimental animals and
human subjects (4, 10-13, 15, 18, 19, 26, 33-37, 41, 42, 44). Clinical trials using rAAV vectors are ongoing for the treatment of cystic fibrosis, hemophilia B, and limb girdle muscular dystrophy (11, 18, 39). Although rAAV vectors have been shown to result in therapeutic and long-term transgene expression when
delivered directly to skeletal muscle and liver in vivo (4, 12,
13, 15, 26, 34-37, 41, 42), the mechanisms by which double-stranded (ds) rAAV vector genomes are stably maintained and
persistently express transgenes in transduced livers have not been
clearly delineated. Since rAAV vectors are capable of integration into
the host chromosomes of mammalian cells in vitro (7, 9, 31, 32,
45), chromosomal integration represents one possible mechanism
by which these vectors maintain persistent transgene expression. In the
context of the muscle, however, in vivo integration has never been
definitively established. Moreover, recent studies suggest that
extrachromosomal ds circular rAAV genomes, which are often referred to
as episomal circular genomes, are likely responsible for long-term
transgene expression in this target tissue (8, 21, 46).
However, we and others have shown that within the liver, rAAV vectors
can integrate into chromosomal DNA in vivo, as determined by
pulsed-field gel electrophoresis, fluorescent in situ hybridization
(FISH), isolation of vector-cellular DNA junction fragments, and in
vivo selection of hepatocytes with integrated rAAV vector genomes
(5, 25, 27). Although extrachromosomal rAAV genomes have
been detected in transduced mouse and dog livers (27, 37,
42; H. Nakai and M. A. Kay, unpublished results), the
proportion of integrated and extrachromosomal genomes responsible for
transgene expression has not been established. The plasmid rescue
method used to demonstrate extrachromosomal circular rAAV genomes in
transduced animal tissues (8, 27) does not allow an
estimation of the relative abundance of the different rAAV genome forms
(e.g., monomeric or concatemeric extrachromosomes or integrated forms)
because not all can be rescued in bacteria. Southern blot analysis has
been used to distinguish different rAAV forms, but it is not able to
distinguish between integrated and extrachromosomal genomes when they
form large concatemers.
To establish the proportion of extrachromosomal and integrated gene
expression and determine the major source of transgene products, we
stimulated hepatocyte division by a surgical two-thirds partial
hepatectomy in rAAV-injected mice at a time when the transgene expression had reached a plateau. Liver cell mass is restored by one or
two cell divisions from each remaining hepatocyte within weeks after
this surgical procedure (17). Thus, surgical hepatectomy generates a condition under which extrachromosomal genomes will be
diluted or lost with cell division, while integrated genomes should not
be diluted, leading to complete restoration during hepatocyte repopulation.
Construction of plasmids and rAAV vectors.
All rAAV vectors
used in this study were based on AAV type 2 and produced by the
triple-transfection method (22). Vectors were purified by
two cycles of cesium chloride gradient ultracentrifugation followed by
ultrafiltration-diafiltration, as previously described (3). The physical particle titers were determined by a
quantitative dot blot assay (19). We used "p" and
"AAV-" as a general system of nomenclature for our plasmids and
rAAV vectors, respectively.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.6969-6976.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Extrachromosomal Recombinant Adeno-Associated Virus Vector
Genomes Are Primarily Responsible for Stable Liver Transduction
In Vivo
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-F.IX, AAV-CM1, and AAV-CM2 were produced based on the
plasmids pV4.1e-hF.IX (26), pAAV-CM1, and pAAV-CM2. To
construct pAAV-CM1, the whole EF1-hF.IX cassette (human elongation
factor 1 [EF1] gene enhancer-promoter-driven human coagulation
factor IX [hF.IX] expression cassette) between two NotI
sites that were located at the inner ends of both inverted terminal
repeats (ITRs) was removed and replaced with another hF.IX expression
cassette from pBS-ApoEHCR(s)-hAATp-hF.IXmg-bp (25), which
contains the apolipoprotein E hepatic locus control region (HCR)-human
1-antitrypsin gene promoter [ApoEHCR(s)-hAATp]-driven hF.IX
minigene (containing a 1.4-kb intron A from the hF.IX gene) with the
bovine growth hormone gene polyadenylation signal. To construct
pAAV-CM2, the hF.IX minigene and polyadenylation signal in pAAV-CM1
were replaced with the hF.IX minigene carrying an 0.3-kb intron A and
3' untranslated region that included the hF.IX polyadenylation signal.
AAV-EF1-F.IX.PMT was produced using 293PMT cells that constitutively
express the bacterial PaeR7 methyltransferase (28). All
the adenine residues at the XhoI recognition sites in
AAV-EF1-F.IX.PMT vector were methylated by the PaeR7
methyltransferase (28, 29). Because our previous studies
showed that most of the ds vector genomes in transduced livers were
formed by annealing of complementary ss genomes (28), ds
AAV-EF1-F.IX.PMT vector genomes were fully adenine methylated at the
XhoI sites and not cleaved with XhoI (which only
cleaves fully unmethylated XhoI sites) unless the ds vector
genomes fully replicated by passing through two or more cell cycles.
Thus, methylation status at the XhoI site serves as a marker
for vector genome replication.
-hF.IX, carrying the EF1 enhancer-promoter-driven hF.IX
expression cassette, and the pCMV-SB and pCMV-mSB transposase plasmids,
encoding wild-type and nonfunctional mutant SB transposase genes,
respectively, under the control of the human cytomegalovirus
immediate-early gene enhancer-promoter, have been described elsewhere
(14, 47).
All plasmids were amplified in either DH10B (Gibco BRL, Gaithersburg,
Md.) or Sure (Stratagene, Cedar Creek, Tex.) bacteria.
Animal procedures.
Six- to eight-week-old female C57BL/6
mice were obtained from either Jackson Laboratory (Bar Harbor, Maine)
or Taconic (Germantown, N.Y.). All animal procedures were performed
according to the guidelines for animal care at Stanford University.
Portal vein injection of rAAV vectors, hydrodynamics-based in vivo
hepatocyte transfection of naked plasmid DNA by tail vein injection,
and partial hepatectomy were performed as previously described
(16, 20, 26, 47, 48). Blood samples were collected from
the retro-orbital plexus. The summary of animal experiments is shown in
Table 1.
|
Measurement of hF.IX in samples. Enzyme-linked immunosorbent assay specific for hF.IX was employed for the measurement of hF.IX in mouse serum or plasma samples using affinity-purified plasma hF.IX (Calbiochem, La Jolla, Calif.) as a standard (40).
Southern blot analysis.
Total genomic DNA was extracted from
livers and subjected to Southern blot analysis as previously described
(27, 28). Briefly, 20 µg of total genomic liver DNAs was
digested with a restriction enzyme or a combination of enzymes,
separated on an 0.8% agarose gel, transferred to a nylon membrane
(Duralon UV; Stratagene), and hybridized with
32P-labeled vector sequence-specific probes.
Membranes were exposed to X-ray films at 
80°C for 3 days to 2 weeks. The vector genome copy number standards (the number of ds vector
genomes per diploid genomic equivalent) were 20 µg of naive mouse
total liver DNAs mixed with the appropriate number of each vector
plasmid. Band intensities were quantitated using a G710 calibrated
imaging densitometer (Bio-Rad, Hercules, Calif.).
-F.IX.PMT-injected mice by quantitative Southern
blot analysis.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Extrachromosomal ds circular monomers are maintained in mouse liver
for over 1.5 years.
To estimate the abundance of extrachromosomal
circular monomer rAAV vector genomes in mouse livers, we injected each
of four mice with 2.7 × 1011 particles of
AAV-EF1-F.IX (Fig. 1) via the portal
vein (Table 1, group 1). Animals were sacrificed at 3, 10, and 19 months postinjection (n = 1, 1, and 2, respectively)
and analyzed by Southern blotting. Our previous study showed the
presence of extrachromosomal vector forms in mouse liver samples
harvested 3 months postinjection (27). At one time, we
thought that these extrachromosomal forms represented ss rAAV genomes
that annealed during the isolation of tissue DNA and not bona fide
molecular forms of the vector DNA (27). However,
subsequent analyses demonstrated that these extrachromosomal bands
primarily represented ds circular forms (as either supercoiled or
relaxed) and/or linear ds monomer genomes (28) that
persist in vivo. The relaxed circular monomer forms may be nicked
supercoiled ds circular forms. In the samples from animals isolated 10 and 19 months postinjection, extrachromosomal forms were readily
detectable by Southern blotting (Fig. 2).
The persistence of extrachromosomal circular rAAV genomes in mouse liver detectable by Southern blotting has been recently reported by
Song et al. (37). All of these data suggest that
considerable amounts of extrachromosomal rAAV vector genomes can
persist in mouse liver.
|
|
Transgene expression from integrated plasmid vector genomes is not
affected by partial hepatectomy.
The Southern blot data shown here
and our previous studies (25, 27) demonstrate the
long-term presence of both integrated and extrachromosomal rAAV
genomes. However, these earlier studies did not establish the relative
proportion of integrated and extrachromosomal genomes or the
contribution of each to transgene expression. In order to establish an
assay that would enable us to distinguish between transgene expression
from integrated genomes and that from extrachromosomal genomes, we
first generated mice that contained an EF1-hF.IX expression cassette
integrated into the chromosomes of mouse hepatocytes, using the SB
transposon system (47). These animals served as a control
for chromosomal integration (Fig. 3;
n = 10, group 2). Mice containing stably transfected
hepatocytes were readily generated by injecting them via the tail vein
with a plasmid containing an hF.IX transposon (pT-EF1-hF.IX [Fig. 1]) together with a helper plasmid encoding the SB transposase (pCMV-SB). Importantly, the expression cassette contained within pT-EF1-hF.IX was identical to one encoded by the rAAV vector AAV-EF1-F.IX. Control groups received pT-EF1-hF.IX either alone (n = 5, group 4) or in combination with pCMV-mSB
(n = 10, group 3), which encodes a catalytically
inactive mutant form of the SB transposase. Since pT-EF1-hF.IX alone
or in combination with pCMV-mSB did not integrate into chromosomes in
hepatocytes in vivo, pT-EF1-hF.IX DNA remained extrachromosomal and
transgene expression was lost within 3 weeks in groups 3 and 4 (Fig.
3). In contrast, stable plasma hF.IX levels of approximately 300 ng/ml were achieved in mice 5 weeks postinjection with pT-EF1-hF.IX and
pCMV-SB (group 2). Thus, virtually all the hF.IX expression observed
after 3 weeks came from integrated transposon genomes.
|
-hF.IX expression cassettes were not diluted and that transgene
expression was not hampered by cell cycling. In addition, this also
showed that 3 weeks was sufficient for a regenerating liver to
completely restore its capacity to express the same levels of hF.IX as
before hepatectomy. These results support partial hepatectomy as a
useful and reasonable strategy to differentiate between the expression from integrated genomes and that from extrachromosomal vector genomes.
Loss of rAAV transgene expression and vector genomes in replicating
hepatocytes.
We previously reported that rAAV-injected mice that
had not yet reached a steady-state level of transgene expression during the first 3 weeks postinjection had a falloff in rAAV genome copy number and transgene expression following partial hepatectomy (24). However, at this time, the rAAV genomes may not have
fully integrated or formed stable structures. Therefore, we examined mice that were stably transduced by rAAV vectors (i.e., 6 or more weeks
postinjection). For these studies, three hF.IX-expressing rAAV vectors were used. Eight mice were injected with either
AAV-CM1 (Fig. 1) or AAV-CM2 (Fig. 1) (n = 4 each,
groups 5 and 6, respectively) at a dose of 3.0 × 1011 vector genomes (vg) per mouse, and two mice
from each group were partially hepatectomized 12 months
postinjection. The serum hF.IX levels at the time of partial
hepatectomy were 8,411 ± 2,687 ng/ml (mean ± standard
deviation) and 6,423 ± 922 ng/ml in AAV-CM1- and AAV-CM2-injected
mice, respectively. We were unable to remove two-thirds of the liver in
one mouse injected with AAV-CM1 because of severe adhesions in the
peritoneal cavity. This animal was sacrificed at the time of surgery.
All the mice were sacrificed 6 weeks after partial hepatectomy, and
their livers were harvested to quantify the vector genomes in the
liver. The serum hF.IX levels relative to those at hepatectomy are
summarized in Fig. 4. The hF.IX levels
dropped by 83 to 89% in hepatectomized mice but remained stable in all
the nonhepatectomized mice. The numbers of vector genomes per cell
before and after hepatectomy were 1.00 versus 0.06 in an AAV-CM1
injected mouse and 1.95 versus 0.63 and 2.18 versus 0.20 in
AAV-CM2-injected mice (Table 2, groups 5 and 6).
|
|
-F.IX (group 7), and half of
these animals were partially hepatectomized 12 weeks postinjection.
Plasma hF.IX levels at the time of partial hepatectomy in the
hepatectomized subgroup and levels in the nonhepatectomized
subgroup were 1,383 ± 214 and 1,597 ± 690 ng/ml,
respectively. As shown in Fig. 5A, the
average hF.IX levels decreased by 95% over a period
of 6 weeks after partial hepatectomy (from 1,383 ± 214 to 71 ± 42 ng/ml) (n = 9; one mouse was sacrificed before
the end point of this study), while the hF.IX levels in the
nonhepatectomized subgroup remained relatively stable during the same
period (1,516 ± 872 ng/ml, corresponding to 6 weeks
posthepatectomy; n = 10). Concordant with the plasma
hF.IX levels, the average number of vector genomes per cell also fell
after partial hepatectomy from 1.02 ± 0.52 copies per cell at the
time of hepatectomy to 0.12 ± 0.03 copy per cell 6 weeks
posthepatectomy (Fig. 5B and C).
|
-F.IX.PMT vector via the portal vein. Twelve months
postinjection, one animal receiving each dose was partially
hepatectomized. The ds vector genomes in mouse livers harvested both at
the time of partial hepatectomy and 3 weeks later were analyzed by
Southern blotting for vector copy number and the presence of
XhoI-digestible (replicated) genomes after digestion with
BglII and in combination with XhoI, respectively. As summarized in Table 2, in a mouse that received 2.4 × 1011 vg (mouse 3), the vector copy number
decreased 44% after partial hepatectomy while 20% of the genomes were
XhoI digestible. The decrease in vector copy number after
hepatectomy was not as dramatic in this mouse as for others in the
previous experiment. In contrast, a mouse that received 7.2 × 1011 vg (mouse 6) showed only a 14% decrease in
vector copy number after hepatectomy, and 67% of the genomes were
digested with XhoI (Fig. 6).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
There is strong evidence that some proportion of rAAV vectors integrate into chromosomes in hepatocytes in vivo (5, 23, 27); however, a series of studies including the present one have raised the importance of extrachromosomal genomes for persistent transgene expression. The relatively small number of integrated genomes clouds the possibility that a single administration of vector will persist lifelong in humans. Additional preclinical studies with large animals will be needed to help clarify this important issue.
After rAAV vectors enter the nuclei of hepatocytes, complementary ss rAAV genomes anneal to form transcriptionally active ds genomes. The ds forms may be converted into large concatemers via random end joining and/or integrate into chromosomes (28). Extrachromosomal ds vector genomes are present as linear and supercoiled circular monomers, as well as both circular and linear concatemers (8, 28, 37, 38). Among these extrachromosomal forms, ds circular monomers and concatemers seem to be the most important genomic structures contributing to persistent gene expression in muscle (8, 46). The fact that the relative fall in rAAV-mediated gene expression was similar in mice that underwent a partial hepatectomy 12 weeks after vector administration and those that underwent a partial hepatectomy 12 months after vector administration suggests that the relative number of extrachromosomal and integrated genomes did not substantially change over this period. However, the reason for the high proportion of integrated genomes in a mouse that received a higher dose of vector is not clear. Further studies are required to establish the kinetics of genome conversion in vivo.
We have previously suggested that the majority of rAAV genomes integrate as head-to-tail concatemers into chromosomes in mouse hepatocytes in vivo after portal vein injection of vector (23). This was based on finding rAAV FISH signals on sister chromatids of metaphase spreads from isolated hepatocytes, as well as a series of molecular analyses that included pulsed-field gel electrophoresis to look at the genome-sized DNA fragments containing rAAV genomes (23). While these previous studies provided us with the first evidence of in vivo integration, our further analyses elucidated that we had overlooked the presence of persistent extrachromosomal rAAV genomes in transduced hepatocytes (28). Our more recent studies of rAAV genome forms in transduced mouse liver in vivo demonstrated the presence of head-to-tail, head-to-head, and tail-to-tail high-molecular-weight rAAV concatemers (either integrated or extrachromosomal), as well as low-molecular-weight extrachromosomal genomes dominated by ds circular monomers. Recent evidence suggests that extrachromosomal FISH signals can associate with chromosomes in metaphase spreads (1). Thus, a certain proportion of DNAs in hepatocyte metaphases might have in fact represented extrachromosomal genomes that tightly associated with chromosomes, overestimating the integration efficiency in these previous studies. The present study clearly demonstrates that many rAAV genomes remain as extrachromosomes in the liver and that these extrachromosomal forms, rather than integrated genomes, are primarily responsible for persistent transgene expression from rAAV vectors.
The mechanistic reasons for the persistence of extrachromosomal rAAV
genomes are not known. Gene expression from exogenous supercoiled
circular plasmid vectors delivered into hepatocytes in vivo is
generally transient, and when persistent, the expression levels are
relatively low (2, 43, 49). Because both plasmids and ds
circular rAAV genomes have similarity in their structures in that they
are both supercoiled circular DNAs residing in nuclei, some unknown
mechanism may operate for ds extrachromosomal rAAV genomes to persist
and continue to express their transgene product. Although we do not yet
understand why gene expression can persist in vivo from
extrachromosomal rAAV genomes, there is some evidence that vector
genome concatemerization and/or the presence of the AAV-ITR sequence
itself may stabilize extrachromosomal genomes in nuclei (8,
30). Recently, Chen et al. reported that persistent, high-level
transgene expression from plasmid-based vectors in mouse hepatocytes
correlated with concatemerization of input linear plasmid vector
genomes (6). On the other hand, Miao et al. have found
that inclusion of the apolipoprotein E HCR together with a part of the
hF.IX gene that included a portion of the first intron greatly
augmented and stabilized hF.IX expression from extrachromosomal
supercoiled monomer plasmid vectors transfected in mouse
hepatocytes in vivo (25). The hypothesis was that a matrix
attachment region included in the HCR and intron sequence may have
facilitated interaction with the nuclear matrix, leading to persistent
gene expression. Since AAV-CM1 and AAV-CM2 contained these elements and
AAV-EF1-F.IX carried an intron, it is also possible that inclusion
of these stabilizing elements rather than concatemerization or the
presence of AAV-ITR may have somehow influenced the persistence of
vector genomes and rAAV-mediated gene expression. However, we have no
evidence to support this assumption. Further analysis of
extrachromosomal rAAV genomes in transduced hepatocytes should help
unravel the mechanisms underlying persistent expression from rAAV genomes.
In summary, our current study establishes an important role for extrachromosomal rAAV vector genomes in maintaining persistent transgene expression in hepatocytes transduced by rAAV vectors. Our finding that the frequency of rAAV genome integration into hepatic chromosomes in vivo is actually quite low further reduces the theoretical risk of harmful side effects incurred during the use of rAAV and other integrating vector systems.
| |
ACKNOWLEDGMENT |
|---|
This work was supported by NIH grant HL64274.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Departments of Pediatrics and Genetics, 300 Pasteur Dr., Room G305A, Stanford University, Stanford, CA 94305. Phone: (650) 498-6531. Fax: (650) 498-6540. E-mail: markay{at}stanford.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Baiker, A., C. Maercker, C. Piechaczek, S. B. Schmidt, J. Bode, C. Benham, and H. J. Lipps. 2000. Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix. Nat. Cell Biol. 2:182-184[CrossRef][Medline]. |
| 2. | Budker, V., G. Zhang, S. Knechtle, and J. A. Wolff. 1996. Naked DNA delivered intraportally expresses efficiently in hepatocytes. Gene Ther. 3:593-598[Medline]. |
| 3. |
Burton, M.,
H. Nakai,
P. Colosi,
J. Cunningham,
R. Mitchell, and L. Couto.
1999.
Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.
Proc. Natl. Acad. Sci. USA
96:12725-12730 |
| 4. | Chao, H., R. Samulski, D. Bellinger, P. Monahan, T. Nichols, and C. Walsh. 1999. Persistent expression of canine factor IX in hemophilia B canines. Gene Ther. 6:1695-1704[CrossRef][Medline]. |
| 5. | Chen, S. J., J. Tazelaar, A. D. Moscioni, and J. M. Wilson. 2000. In vivo selection of hepatocytes transduced with adeno-associated viral vectors. Mol. Ther. 1:414-422[CrossRef][Medline]. |
| 6. | Chen, Z.-Y., S. Yant, C.-Y. He, L. Meuse, S. Shen, and M. A. Kay. 2000. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol. Ther. 3:403-410. |
| 7. | Duan, D., K. J. Fisher, J. F. Burda, and J. F. Engelhardt. 1997. Structural and functional heterogeneity of integrated recombinant AAV genomes. Virus Res. 48:41-56[CrossRef][Medline]. |
| 8. |
Duan, D.,
P. Sharma,
J. Yang,
Y. Yue,
L. Dudus,
Y. Zhang,
K. J. Fisher, and J. F. Engelhardt.
1998.
Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue.
J. Virol.
72:8568-8577 |
| 9. |
Fisher-Adams, G.,
K. K. Wong, Jr.,
G. Podsakoff,
S. J. Forman, and S. Chatterjee.
1996.
Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.
Blood
88:492-504 |
| 10. |
Flotte, T. R.,
S. A. Afione,
C. Conrad,
S. A. McGrath,
R. Solow,
H. Oka,
P. L. Zeitlin,
W. B. Guggino, and B. J. Carter.
1993.
Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.
Proc. Natl. Acad. Sci. USA
90:10613-10617 |
| 11. | Greelish, J. P., L. T. Su, E. B. Lankford, J. M. Burkman, H. Chen, S. K. Konig, I. M. Mercier, P. R. Desjardins, M. A. Mitchell, X. G. Zheng, J. Leferovich, G. P. Gao, R. J. Balice-Gordon, J. M. Wilson, and H. H. Stedman. 1999. Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector. Nat. Med. 5:439-443[CrossRef][Medline]. |
| 12. |
Herzog, R. W.,
J. N. Hagstrom,
S. H. Kung,
S. J. Tai,
J. M. Wilson,
K. J. Fisher, and K. A. High.
1997.
Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus.
Proc. Natl. Acad. Sci. USA
94:5804-5809 |
| 13. | Herzog, R. W., E. Y. Yang, L. B. Couto, J. N. Hagstrom, D. Elwell, P. A. Fields, M. Burton, D. A. Bellinger, M. S. Read, K. M. Brinkhous, G. M. Podsakoff, T. C. Nichols, G. J. Kurtzman, and K. A. High. 1999. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat. Med. 5:56-63[CrossRef][Medline]. |
| 14. | Ivics, Z., P. B. Hackett, R. H. Plasterk, and Z. Izsvak. 1997. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91:501-510[CrossRef][Medline]. |
| 15. | Kaplitt, M. G., P. Leone, R. J. Samulski, X. Xiao, D. W. Pfaff, K. L. O'Malley, and M. J. During. 1994. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat. Genet. 8:148-154[CrossRef][Medline]. |
| 16. | Kay, M. A., Q. Li, T. J. Liu, F. Leland, C. Toman, M. Finegold, and S. L. Woo. 1992. Hepatic gene therapy: persistent expression of human alpha 1-antitrypsin in mice after direct gene delivery in vivo. Hum. Gene Ther. 3:641-647[Medline]. |
| 17. | Kay, M. A., and N. Fausto. 1997. Liver regeneration: prospects for therapy based on new technologies. Mol. Med. Today 3:108-115[CrossRef][Medline]. |
| 18. | Kay, M. A., C. S. Manno, M. V. Ragni, P. J. Larson, L. B. Couto, A. McClelland, B. Glader, A. J. Chew, S. J. Tai, R. W. Herzog, V. Arruda, F. Johnson, C. Scallan, E. Skarsgard, A. W. Flake, and K. A. High. 2000. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24:257-261[CrossRef][Medline]. |
| 19. |
Kessler, P. D.,
G. M. Podsakoff,
X. Chen,
S. A. McQuiston,
P. C. Colosi,
L. A. Matelis,
G. J. Kurtzman, and B. J. Byrne.
1996.
Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.
Proc. Natl. Acad. Sci. USA
93:14082-14087 |
| 20. | Liu, F., Y. Song, and D. Liu. 1999. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6:1258-1266[CrossRef][Medline]. |
| 21. |
Malik, A. K.,
P. E. Monahan,
D. L. Allen,
B. G. Chen,
R. J. Samulski, and K. Kurachi.
2000.
Kinetics of recombinant adeno-associated virus-mediated gene transfer.
J. Virol.
74:3555-3565 |
| 22. | Matsushita, T., S. Elliger, C. Elliger, G. Podsakoff, L. Villarreal, G. J. Kurtzman, Y. Iwaki, and P. Colosi. 1998. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther. 5:938-945[CrossRef][Medline]. |
| 23. | Miao, C. H., R. O. Snyder, D. B. Schowalter, G. A. Patijn, B. Donahue, B. Winther, and M. A. Kay. 1998. The kinetics of rAAV integration in the liver. Nat. Genet. 19:13-15[CrossRef][Medline]. |
| 24. |
Miao, C. H.,
H. Nakai,
A. R. Thompson,
T. A. Storm,
W. Chiu,
R. O. Snyder, and M. A. Kay.
2000.
Nonrandom transduction of recombinant adeno-associated viral vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction.
J. Virol.
74:3793-3803 |
| 25. | Miao, C. H., K. Ohashi, G. A. Patijn, L. Meuse, X. Ye, A. R. Thompson, and M. A. Kay. 2000. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol. Ther. 1:522-532[CrossRef][Medline]. |
| 26. |
Nakai, H.,
R. W. Herzog,
J. N. Hagstrom,
J. Walter,
S. H. Kung,
E. Y. Yang,
S. J. Tai,
Y. Iwaki,
G. J. Kurtzman,
K. J. Fisher,
P. Colosi,
L. B. Couto, and K. A. High.
1998.
Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver.
Blood
91:4600-4607 |
| 27. |
Nakai, H.,
Y. Iwaki,
M. A. Kay, and L. B. Couto.
1999.
Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver.
J. Virol.
73:5438-5447 |
| 28. |
Nakai, H.,
T. A. Storm, and M. A. Kay.
2000.
Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.
J. Virol.
74:9451-9463 |
| 29. | Nelson, J. E., and M. A. Kay. 1997. Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication. J. Virol. 71:8902-8907[Abstract]. |
| 30. |
Philip, R.,
E. Brunette,
L. Kilinski,
D. Murugesh,
M. A. McNally,
K. Ucar,
J. Rosenblatt,
T. B. Okarma, and J. S. Lebkowski.
1994.
Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.
Mol. Cell. Biol.
14:2411-2418 |
| 31. | Ponnazhagan, S., D. Erikson, W. G. Kearns, S. Z. Zhou, P. Nahreini, X. S. Wang, and A. Srivastava. 1997. Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells. Hum. Gene Ther. 8:275-284[Medline]. |
| 32. | Rutledge, E. A., and D. W. Russell. 1997. Adeno-associated virus vector integration junctions. J. Virol. 71:8429-8436[Abstract]. |
| 33. | Skorupa, A. F., K. J. Fisher, J. M. Wilson, M. K. Parente, and J. H. Wolfe. 1999. Sustained production of beta-glucuronidase from localized sites after AAV vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice. Exp. Neurol. 160:17-27[CrossRef][Medline]. |
| 34. | Snyder, R. O., C. H. Miao, G. A. Patijn, S. K. Spratt, O. Danos, D. Nagy, A. M. Gown, B. Winther, L. Meuse, L. K. Cohen, A. R. Thompson, and M. A. Kay. 1997. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat. Genet. 16:270-276[CrossRef][Medline]. |
| 35. | Snyder, R. O., S. K. Spratt, C. Lagarde, D. Bohl, B. Kaspar, B. Sloan, L. K. Cohen, and O. Danos. 1997. Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. Hum. Gene Ther. 8:1891-1900[Medline]. |
| 36. | Snyder, R. O., C. Miao, L. Meuse, J. Tubb, B. A. Donahue, H. F. Lin, D. W. Stafford, S. Patel, A. R. Thompson, T. Nichols, M. S. Read, D. A. Bellinger, K. M. Brinkhous, and M. A. Kay. 1999. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat. Med. 5:64-70[CrossRef][Medline]. |
| 37. | Song, S., J. Embury, P. J. Laipis, K. I. Berns, J. M. Crawford, and T. R. Flotte. Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors. Gene Ther., in press. |
| 38. |
Vincent-Lacaze, N.,
R. O. Snyder,
R. Gluzman,
D. Bohl,
C. Lagarde, and O. Danos.
1999.
Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle.
J. Virol.
73:1949-1955 |
| 39. | Wagner, J. A., A. H. Messner, M. L. Moran, R. Daifuku, K. Kouyama, J. K. Desch, S. Manley, A. M. Norbash, C. K. Conrad, S. Friborg, T. Reynolds, W. B. Guggino, R. B. Moss, B. J. Carter, J. J. Wine, T. R. Flotte, and P. Gardner. 1999. Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope 109:266-274[CrossRef][Medline]. |
| 40. |
Walter, J.,
Q. You,
J. N. Hagstrom,
M. Sands, and K. A. High.
1996.
Successful expression of human factor IX following repeat administration of adenoviral vector in mice.
Proc. Natl. Acad. Sci. USA
93:3056-3061 |
| 41. | Wang, L., T. C. Nichols, M. S. Read, D. A. Bellinger, and I. M. Verma. 2000. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol. Ther. 1:154-158[CrossRef][Medline]. |
| 42. |
Wang, L.,
K. Takabe,
S. M. Bidlingmaier,
C. R. Ill, and I. M. Verma.
1999.
Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.
Proc. Natl. Acad. Sci. USA
96:3906-3910 |
| 43. |
Wu, C. H.,
J. M. Wilson, and G. Y. Wu.
1989.
Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo.
J. Biol. Chem.
264:16985-16987 |
| 44. | Xiao, X., J. Li, and R. J. Samulski. 1996. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J. Virol. 70:8098-8108[Abstract]. |
| 45. | Yang, C. C., X. Xiao, X. Zhu, D. C. Ansardi, N. D. Epstein, M. R. Frey, A. G. Matera, and R. J. Samulski. 1997. Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J. Virol. 71:9231-9247[Abstract]. |
| 46. |
Yang, J.,
W. Zhou,
Y. Zhang,
T. Zidon,
T. Ritchie, and J. F. Engelhardt.
1999.
Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination.
J. Virol.
73:9468-9477 |
| 47. | Yant, S. R., L. Meuse, W. Chiu, Z. Ivics, Z. Izsvak, and M. A. Kay. 2000. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat. Genet. 25:35-41[CrossRef][Medline]. |
| 48. | Zhang, G., V. Budker, and J. A. Wolff. 1999. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther. 10:1735-1737[CrossRef][Medline]. |
| 49. | Zhang, G., D. Vargo, V. Budker, N. Armstrong, S. Knechtle, and J. A. Wolff. 1997. Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers. Hum. Gene Ther. 8:1763-1772[Medline]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
