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J Virol, June 1998, p. 5025-5034, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Site-Specific Integration in Mammalian Cells Mediated by a New
Hybrid Baculovirus-Adeno-Associated Virus Vector
Fabio
Palombo,
Andrea
Monciotti,
Alessandra
Recchia,
Riccardo
Cortese,
Gennaro
Ciliberto, and
Nicola
La
Monica*
IRBM P. Angeletti, 00040 Pomezia, Italy
Received 5 November 1997/Accepted 2 March 1998
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ABSTRACT |
Baculovirus can transiently transduce primary human and rat
hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the
elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the 
-galactosidase
(-Gal) reporter gene and the hygromycin resistance
(Hygr) gene flanked by the AAV inverted terminal repeats
(ITRs), which are necessary for AAV replication and integration in
the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different
positions with respect to the baculovirus polyheidrin promoter. A
high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus
containing the ITR-Hygr--Gal sequence was obtained
with insect cells only when the rep gene was placed in an
antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in
a 10- to 50-fold increase in the number of Hygr stable cell
clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in
approximately 41% of cases as detected by both Southern blotting and
fluorescent in situ hybridization analysis. Moreover, site-specific
integration of the ITR-flanked DNA was also detected by PCR
amplification of the ITR-aavs1 junction in transduced human
fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow
permanent, nontoxic gene delivery of DNA constructs for ex vivo
treatment of primary human cells.
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INTRODUCTION |
Gene therapy is a rapidly emerging
field that aims to treat a variety of genetic or acquired diseases
through the transfer of functional genetic material into cells both in
vitro and in vivo (1, 30, 34). Critical to the success of
gene therapy is the development of safe and efficient gene transfer
vehicles. To date, various strategies have been developed for the
transfer of therapeutic genes which include viral and nonviral vectors. All of these gene delivery systems, however, suffer from limitations in
their applicability and efficacy (49). Among the viral
vectors utilized for gene transfer protocols, adenovirus (Ad) vectors deliver genes to a wide variety of cell types and tissues independently of their proliferative state (9). However, the major
disadvantage of this type of vector is the instability of the genes
transferred into the target cell and the substantial pathology that
develops at the site of gene transfer. These problems can be explained, at least in part, by a lack of integration of the recombinant Ad genome
and by the development of a strong cellular immune response to the
genetically corrected cells that express low levels of viral proteins
(13, 53). Despite some improvements in reducing the
immunogenicity of Ad vectors (3, 50, 55), their use for
long-term expression of the therapeutic gene has yet to be fully
assessed.
Retroviruses, the viral vectors currently most widely used, offer the
desirable feature of being able to insert a gene of interest into the
host genome, thus contributing to the stability of the transduced gene
(12). However, retroviruses have a limited host range, and
successful infection occurs only in mitotic cells, with the exception
of the human immunodeficiency virus (31). Additionally,
retroviruses integrate randomly into the host cell chromosome, thus
raising some concern about the potential activation of
transcriptionally silent oncogenes and the possible inactivation of
tumor suppressor genes mediated by insertional mutagenesis (43).
The adeno-associated virus (AAV) is also used for gene delivery
protocols. The lack of obvious pathogenic effects associated with AAV
infection and the stability of the viral particle have elicited great
interest in the use of AAV as a vector in the field of gene therapy
(10). Additionally, AAV is the only known eukaryotic virus
that preferentially integrates its DNA into a defined region of the
host cell genome located on chromosome 19q13.3 (16, 28, 40).
Although the mechanism of site-specific integration is still
unclear, two viral elements, namely, the inverted terminal repeats
(ITRs) and the Rep polypeptides, are required for targeting and
inserting the viral DNA into the integration locus aavs1
(42). The two 145-nucleotide ITRs located at both ends of
the viral genome are the minimal cis-acting sequences
required in the integration process. The palindromic portions of the
ITRs are capable of forming hairpin structures and serve as a
self-priming origin for DNA replication, as well as a site for binding
of AAV Rep proteins (20, 44, 48). The rep gene is
transcribed from two promoters, p5 and p19. Transcription from the p5
promoter generates spliced and unspliced mRNAs which encode the
Rep68 and Rep78 proteins, respectively. Two smaller proteins, Rep40 and
Rep52, are encoded by spliced and unspliced mRNAs promoted by the
p19 promoter, respectively (45). These proteins mediate
replication and integration of the AAV genome, as well as packaging
into viral particles (7). In particular, Rep68 and Rep78
interact with the terminal sequences only if the secondary structure of
the termini is T shaped, a conformation that is believed to exist in
virion DNA (4, 20). Additionally, these two large Rep
polypeptides contain ATP-dependent helicase and strand-specific
endonuclease activities which are thought to be important both for
viral DNA replication and integration (21, 22). Furthermore,
recent evidence has indicated that site-specific integration of an
ITR-flanked DNA segment requires expression of the Rep proteins
(5, 37) and that with only the expression of Rep78 or Rep68,
highly efficient integration into the aavs1 site is obtained
(46).
The major limitation of AAV-based vectors resides in the size
constraint imposed by the inability of the mature AAV particles to
package DNA fragments larger than 5 kb. Therefore, recombinant AAV
vectors are generated by substituting the endogenous genes with
exogenous DNA, resulting in the production of vectors that can no
longer integrate in a site-specific manner. In fact, although these
viruses can persist both in vitro and in vivo for prolonged periods of
time, they do so either as episomes or by integrating randomly into the
host chromosome (11, 14, 15, 24-27, 33, 38, 52).
Recently, the use of the Autographa californica multiple
nuclear polyhedrosis virus as a vector for the delivery of genes into
mammalian cells has been reported (8, 19). Although baculoviruses are normally utilized for the overexpression of recombinant proteins in insect cells (35) or as
biopesticides (12), it was shown that A. californica multiple nuclear polyhydrosis virus can infect human
hepatocytes, leading to an efficient transient expression of reporter
genes under the control of an appropriate mammalian promoter (8,
19). In view of the ease of preparation and large cloning
capacity of baculovirus (36), its use as vector for gene
transfer holds great potential. However, both the inability of
baculovirus to transduce cells in vivo (41) and the
transient nature of the transferred DNA pose some limitations for its
use. To determine whether the persistence of the baculovirus-transduced DNA can be prolonged, we introduced the AAV rep gene and a
transgene cassette flanked by the AAV terminal repeats into a
baculovirus backbone. We here show that a stable baculovirus-AAV
(Bac-AAV) hybrid vector can be produced in insect cells at a high titer provided that the rep gene is cloned into an antisense
orientation with respect to the baculovirus polyhedrin promoter.
Additionally, we have determined that Bac-AAV infection of 293 cells
results in highly efficient integration of the transgene cassette in
the aavs1 site. Lastly, we observed that Bac-AAV can efficiently
transduce human diploid fibroblasts, which results in the site-specific integration of ITR-flanked DNA. These findings extend the use of
baculovirus as a vector for gene therapy.
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MATERIALS AND METHODS |
Vector constructs.
Recombinant baculoviruses were
constructed by using the transfer vector pFastBac1 (pFB1) (Gibco-BRL).
A -galactosidase (-Gal)-expressing cassette was derived from
plasmid pCMV- (Clontech). The
EcoRI-HindIII fragment containing the
cytomegalovirus (CMV) promoter, the -Gal gene, and the simian virus
40 (SV40) polyadenylation signal was inserted in the modified sites of
pFB1, generating plasmid pFB1Bac/CMV-. To construct the chimeric
Bac-AAV vectors, a p5-Rep-poly(A) cassette (ClaI and
SpeI) was subcloned into the NspV and
SpeI sites of pFB1, generating pFB1Rep-A.
A transgene cassette was assembled in plasmid pLitmus-28 (New England
Biolabs) by three cloning steps, generating pLit/AAV-Hyg--Gal, which
contains the following sequences: (i) the AAV ITRs derived from plasmid
pSub201 (39) as a PvuII fragment and subcloned in
the blunted XbaI site of pLitmus-28; (ii) the hygromycin
resistance (Hygr) gene excised from plasmid pCEP-4
(Invitrogen) by digestion with NruI and NotI and
inserted between the ITRs in the filled-in XbaI site; and
(iii) the -Gal cassette derived from plasmid CMV-, which was
introduced between the ITRs by blunt-end cloning into the filled-in
NotI and HindIII sites.
Plasmid pLit/AAV-Hyg-
-Gal was digested with
SpeI and
AvrII, and the 6.5-kb fragment containing the transgene
cassette was
subcloned into the modified pFB1 vector (digested with
XbaI and
AvrII) or pFB1RepA to generate Bac-ITR
and Bac/ITR-RepA, respectively.
Similarly, to generate plasmid
Bac-RepS, the p5-Rep fragment was
inserted in the
SfuI site
of pFB1. Bac-ITR/RepS was derived from
Bac-Rep-S by introducing the
transgene cassette as a
AvrII-
SpeI
fragment into
the
SpeI site.
Recombinant baculoviruses were produced according to the
manufacturer's instructions (Gibco-BRL). Viruses were propagated
in
Sf9 insect cells according to standard methods (
36). Budded
virus from insect cell culture medium was filtered on a 0.22-µm
(pore
size) filter and concentrated by ultrafiltration in a 45
Ti rotor
(30,000 rpm, 75 min). The viral pellet was resuspended
in
phosphate-buffered saline (PBS), and the titers of the virus
were
determined by plaque assay on Sf9 insect cells.
Cell culture.
293, Huh-7, and MRC-5 cells were maintained in
Dulbecco modified Eagle medium supplemented with 10% fetal calf serum,
2 mM glutamine, 100 U of penicillin per ml, and 100 µg of
streptomycin per ml. Cells were grown in 10-cm-diameter dishes (Falcon)
at 37°C in 5% CO2. Stock cells were routinely passaged
every 3 days by treatment with trypsin (0.05%) and EDTA (0.53 mM) and
then replated at cell densities appropriate for exponential growth.
To isolate stable cell clones, the infected cells from a single
6-cm-diameter plate were plated at a density of 2 × 10
4 cells per plate in 10 plates containing selection
medium (Dulbecco
modified Eagle medium, 10% fetal calf serum, and 300 µg of hygromycin
per ml). Hygromycin-resistant (Hyg
r)
clones were isolated after 10 days of selection and then expanded
and processed for genomic DNA extraction, Southern blotting, and
fluorescence in situ hybridization (FISH) analysis. Assays for
-Gal
activity were carried out using the
-Gal enzyme assay system
(Promega) as described by the manufacturer.
-Gal was detected
histochemically in cells fixed with 0.5% glutaraldehyde solution
in
PBS and by incubation for 4 h with staining solution (4 mM
K
4[Fe(CN)
6], 4 mM
K
3[Fe(CN)
6], 40 mM MgCl
2,
0.4-mg/ml X-Gal
[5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside]
in
PBS).
Western blot analysis.
The expression of AAV Rep proteins
was evaluated by Western blotting with a polyclonal antibody which
recognizes all four Rep isoforms (47). A total of 2 × 106 Sf9 cells were infected at a multiplicity of infection
(MOI) of 10, and 72 h later cells were collected, washed in PBS,
and lysed by freezing and thawing three times in lysis buffer [10 mM
HEPES buffer [pH 8.0], 0.6 M NaCl]. Portions (200 µg) of proteins of each sample were separated by sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The nitrocellulose membrane was immersed in
1% skimmed milk in Tris-buffered saline (20 mM Tris-HCl, 500 mM NaCl
[pH 7.5]) (blocking buffer) for 20 min at room temperature. The
anti-Rep polyclonal antiserum diluted in blocking buffer was applied to
the nitrocellulose membrane and incubated for 1 h at room
temperature. The membrane was then washed repeatedly with blocking
buffer. The bands were visualized with ECL reagents according to the
manufacturer's instructions (Amersham).
Southern blot analysis.
Baculovirus genomic DNA was prepared
from 1-ml portions of the third viral passage according to standard
protocols (35). One microgram of viral DNA was digested with
EcoRV, which releases two fragments of 4 and 10 kb. After
electrophoresis in 1% agarose gel, the digested fragments were
transferred onto a nylon membrane (Hybond N+), processed according to
the manufacturer's instructions, and hybridized overnight at 65°C in
Church buffer (7% SDS, 0.25 M NaPi [pH 7.2], 1 mM EDTA [pH 8.0],
bovine serum albumin [0.1 g/ml]) with random primed
32P-labeled probes. The following probes were used: for the
gentamicin gene a 1.3-kb EcoRV-BamHI fragment
derived from the plasmid pFastBac1 and for the transgene cassette a
6.5-kb AvrII-SpeI fragment derived from plasmid
pLit/AAV-Hyg--Gal.
To determine site-specific integration of the ITR-DNA fragment, genomic
DNA was prepared as previously described (
37) and
processed
as described above. Filters were first hybridized with
a probe specific
for the transgene cassette; the hybridized probe
was then removed by
boiling the filters in 0.2× SSC (1× SSC is
0.15 M NaCl plus 0.015 M
sodium citrate) and 1% SDS for 10 min,
and the same filters were then
hybridized to a probe covering
nucleotides 1 to 3,525 of aavs1.
FISH.
A 6.5-kb DNA fragment corresponding to the transgene
cassette and an 80-kb aavs1 DNA fragment isolated by screening a
genomic DNA library were labeled by using the Nick Translation Kit
(Boehringer Mannheim) according to the manufacturer's instructions and
used as probes in chromosome analysis.
The chromosome spreads from selected clones were prepared according to
typical cytogenetic techniques (
29). Cytogenetic
preparations were pretreated with pepsin solution and dehydrated
by
washing with cold 70, 90, and 100% ethanol. The preparations
were then
denatured with a 50% formamide solution. For each sample,
200 ng of
probe, 2 µg of human Cot-1 DNA, and 9 µg of sonicated
salmon sperm
DNA were precipitated and resuspended in hybridization
buffer (50%
formamide, 2× SSC, 1% bovine serum albumin, and 10%
dextran
sulfate). Probes were denatured for 8 min at 80°C and
subsequently
incubated for 10 min at 37°C to allow preannealing
of repeated
sequences. Finally, the hybridization solution was
placed on the
samples, covered with coverslips, and incubated
overnight at 37°C
in a moist chamber. The samples were then washed
three times in 50%
formamide and three times in 2× SSC at 42°C.
Visualization of the
biotin-labeled probe was carried out by repeated
incubations with
Cy3-avidin (Amersham), biotinylated anti-avidin
D (Vector
Laboratories), and again with Cy3-avidin. The digoxigenin-labeled
probe
was detected by using mouse anti-digoxigenin antibody,
digoxigenin-labeled
anti-mouse antibody, and fluorescein isothiocyanate
(FITC)-labeled
anti-digoxigenin antibody (Boehringer Mannheim).
Alternatively,
FITC-avidin (Vector Laboratories) and rhodamine-labeled
antidigoxigenin
(Boehringer Mannheim) were used. After immunodetection,
slides
were counterstained with 200 ng of 4',6-diamidino-2-phenylindole
(DAPI). UV excitation was used to locate metaphases, and photographic
images were taken with a charge-coupled-device (CCD) camera
(Photometrics)
with green (FITC) or blue-violet (Cy3 or rhodamine)
illumination.
Images were processed with Adobe Photoshop on an Apple
Quadra
computer.
PCR amplification of the ITR-aavs1 junction.
Integration of
ITR-flanked DNA in the aavs1 site was determined by PCR by using nested
primer pairs that flank the AAV-chromosome junction as previously
described (17, 37): primers 16s (AAV), 5'-GTAGCATGGCGGGTTAATCA, and 15a (aavs1),
5'-GCGCGCATAAGCCAGTAGAGC, were used in the first round of
PCR amplification with 0.5 mg of genomic DNA as the substrate. After an
initial incubation for 4 min at 94°C, the reaction mixture was
subjected to 30 cycles of PCR amplification for 1 min at 94°C, 1 min
at 55°C, and 2 min at 72°C. One percent of the amplification
product was diluted into a new reaction mixture containing a set of
nested primers with the following sequences: 17s (AAV),
5'-TTAACTACAAGGAACCCCTA, and Cr2 (aavs1),
5'-ACAATGGCCAGGGCCAGGCAG. The PCR parameters were the same
as for the first amplification. For molecular cloning of the amplified
junction fragments, the product of the second round of amplification
was purified on a 1% agarose gel and subcloned by blunt end ligation
into plasmid pZERO-2.1 (Invitrogen). Sequencing was performed by
standard chain termination protocols.
| |
RESULTS |
Construction of hybrid Bac-AAV vectors.
To take advantage of
the ease of manipulation of baculovirus and of the unique ability of
AAV virus to preferentially integrate its viral DNA into a defined
region of human chromosome 19, we constructed a series of
baculovirus vectors carrying AAV components known to be required for
excision and integration of the AAV genome in the aavs1 site in human
cells. Figure 1 shows the vectors used in
this study. To determine the transduction efficiency of the recombinant
baculoviruses and to establish stable cell clones from the infected
cells, the -Gal and Hygr genes were inserted between the
AAV ITRs and cloned downstream of the baculovirus polyheidrin promoter
(pPolh) (Bac-ITR). Additionally, the AAV Rep gene under the control of
its own promoters p5 and p19 was cloned outside of the ITRs either in
the sense orientation (Bac-ITR/RepS) or in the antisense orientation
(Bac-ITR/RepA) with respect to the pPolh promoter. Lastly,
baculoviruses carrying the CMV--Gal expression cassette
(Bac--Gal) or the rep gene (Bac-Rep) were also
constructed as controls for transduction efficiency and Rep expression.
Viruses were produced at high titers (ranging from 1 × 109 to 9 × 109 PFU/ml) and used to infect
mammalian cells.
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FIG. 1.
Schematic representation of the recombinant
baculoviruses used in this study. Baculovirus transfer plasmids were
derived from pFastBac1 as described in Materials and Methods. The
E. coli -Gal gene, the Hygr gene (HYGRO), the
rep gene (Rep), and the AAV ITRs are indicated. The
expression of the -Gal and Hygr genes is driven by the
CMV and TK promoters, respectively. The p5 and p19 promoters regulate
expression of the rep gene. Transcription initiation sites
of the baculovirus polyhedrin promoter (pPolh) and of the p5 and p19
promoters are indicated by an arrow. Relevant EcoRV
restriction sites are indicated with an E.
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In an initial functional analysis of the transducing capacity of the
recombinant baculovirus vectors, we observed that the
transient
-Gal
gene expression in infected strain 293 cells generally
increased as a
function of the MOI, a finding in agreement with
published data
(references
8 and
18 and data not shown).
To determine
whether the transducing capacity of baculovirus could
be extended to
other cell types, baculovirus transduction efficiency
in human diploid
fibroblast MRC-5 was compared to that observed
in the human hepatoma
cell line Huh-7. The hepatoma cell line
has been shown to be extremely
susceptible to baculovirus transduction
(
19). Figure
2 shows
-Gal gene expression detected
in MRC-5
and Huh-7 cells infected with Bac-
-Gal at different MOIs
(25,
50, and 100) at 48 h postinfection. These two cell lines
showed
comparable levels of
-Gal, thus indicating that these cells
are
equally susceptible to baculovirus infection; similar results
were
obtained upon infection of primary rat fibroblasts (data
not shown).
These results indicate that baculovirus transducing
capacity can also
be extended to primary cultures that are not
necessarily of hepatic
origin.
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FIG. 2.
Baculovirus-mediated expression of -Gal in MRC-5 and
Huh-7 cells. Cells (105) were plated and infected with
Bac--Gal at the MOI indicated. At 48 h postinfection total
cell extracts were prepared, normalized for protein content, and
assayed for -Gal activity. Each column reflects the average of
results of two independent assays, with the error bars representing the
standard deviations.
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Stability of recombinant Bac-AAV genomes.
When the
transduction efficiencies of Bac-ITR, Bac-ITR/RepA, and
Bac-ITR/RepS were compared by infecting 293 cells at MOIs of 50 and
100, we found that the Bac-ITR virus efficiency was significantly
higher than that of Bac-ITR/RepA. However, both viruses displayed a
similar increase in -Gal gene expression as a function of MOI. In
contrast, very little, if any, -Gal gene expression could be
detected upon infection of 293 cells with Bac-ITR/RepS (Fig.
3).
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FIG. 3.
Baculovirus-mediated expression of -Gal activity in
strain 293 cells infected with different constructs (Bac-ITR,
Bac-ITR/RepS, and Bac-ITR/RepA) at MOIs of 50 and 100. Infection
of 293 cells, preparation of cell extracts, and the -Gal assay were
carried out as indicated in Materials and Methods.
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To determine whether the Bac-ITR/RepS genome had undergone partial
rearrangement during virus amplification, thus explaining
its low
transducing efficiency, we examined the integrity of Bac-AAV
genomes by
Southern blot analysis. Genomic DNA was digested with
EcoRV and resolved on an agarose gel.
EcoRV was
chosen because
it cleaves once within the ITR-flanked transgene
sequence and
once within the gentamicin gene, releasing three fragments
of
10, 4, and 1.3 kb. Figure
4 shows the
viral DNA genomic blots
hybridized with a probe specific for the
transgene cassette (Fig.
4A) and for the gentamicin gene (Fig.
4B). The
gentamicin gene
is present in all virus genomes and therefore was used
as an internal
control. Quantification of transgene probe
hybridization, normalized
for that of gentamicin, indicated that the
hybridization signal
of Bac-ITR/RepA was 11.4-fold higher than that of
Bac-ITR/RepS
(Fig.
4A, compare lane 3 to lane 1), whereas no
significant difference
was observed between Bac-ITR/RepA and Bac-ITR
(Fig.
4A, compare
lane 3 to lane 2). Thus, these data suggest that the
low transduction
efficiency of Bac-ITR/RepS may be ascribed to the loss
or rearrangement
of the ITR-flanked transgene cassette during the
process of baculovirus
amplification.
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FIG. 4.
Stability of recombinant Bac-AAV viruses. (A) Southern
blot analysis of baculovirus genomes. One microgram of viral DNA
prepared from the third viral passage was digested with
EcoRV, fractionated on 1% agarose gel, transferred to a
nylon membrane, and then hybridized with a probe specific for the
transgene sequence. The expected bands of 4 and 10 kb are indicated by
an arrow. (B) The same filter was stripped and hybridized with a probe
specific for the gentamicin gene, which recognizes a 1.3-kb band. The
intensity of the bands (indicated by arrows) in both panels A and B
were quantified with a phosphorimager according to the
manufacturer's instructions. (C) Western blot detection of Rep
isoforms expressed in Sf9 cells infected with different baculoviruses
at an MOI of 10. At 2 days postinfection the total cell extracts were
analyzed for the presence of Rep proteins. Rep78, Rep68, and Rep52 are
indicated by an arrow.
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The three recombinant viral genomes produced in
Escherichia
coli did not show any detectable rearrangement (data not shown);
therefore, we hypothesized that loss of the
-Gal and
Hyg
r expression cassette occurred during Bac-ITR/RepS
amplification
in insect cells. Specifically, in view of the role of the
Rep
polypeptides in promoting the selective excision and amplification
of the ITR-flanked DNA (
44,
48), we speculated that loss of
the transgene cassette could be associated with the expression
of Rep
isoforms in Sf9 cells. To verify this hypothesis, insect
cells were
infected with the Bac-AAV vectors and the Rep expression
pattern was
assessed by Western blotting. As shown in Fig.
4C,
Rep78, Rep68, and
Rep52 could be clearly detected in Bac-Rep infected
cells (Fig.
4C,
lane 1), whereas no Rep polypeptides could be
detected in Bac-ITR cell
lysates (Fig.
4C, lane 2). Interestingly,
the expression level of Rep
polypeptides differed between Bac-ITR/RepS
and Bac-ITR/RepA cell
lysates (Fig.
4C, compare lanes 2 to lanes
4).
rep gene
expression was lower in cells infected with the Bac-AAV
carrying the
rep gene in the antisense orientation with respect
to pPolh
than in the corresponding virus carrying the
rep gene
in a
sense orientation. Thus, in agreement with the Southern blot
analysis
of genomic DNA, these results suggest that the ITR-flanked
cassette is
destabilized in Bac-AAV virus genome when Rep proteins
are expressed
above a certain threshold.
Bac-AAV infection of 293 cells.
Recent studies have shown that
transfection of strain 293 cells with a plasmid carrying the
rep gene and a green fluorescence protein expression
cassette inserted between the AAV terminal repeats results in a highly
efficient integration of the ITR-flanked DNA (5, 37, 46). On
the basis of this observation we wanted to determine whether the
delivery of AAV components mediated by baculovirus can establish more
stable cell clones. To this end, 293 cells were infected with
Bac--Gal, Bac-ITR/RepA, and Bac-ITR vectors, and the transduction
efficiencies of these viruses and the stable integration of the
ITR-flanked DNA were compared by measuring -Gal expression and
the production of Hygr clones.
Figure
5A shows the reporter activity
measured in infected cells cultured in the absence of selection as a
function of time
(up to 14 days postinfection). Although the
transduction efficiencies
determined at day 1 postinfection were
similar with all three
vectors (data not shown), the residual
-Gal
activity at day 14
postinfection was much higher in the
Bac-ITR/RepA-infected cells
than in those infected with either Bac-ITR,
which does not contain
the
rep gene, or Bac-
-Gal, which
contains only the
-Gal expression
cassette.
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FIG. 5.
(A) Time course detection of -Gal activity in strain
293 cells infected with different baculovirus vectors (CMV--Gal,
Bac-ITR, and Bac-ITR/RepA) at an MOI of 100. Reporter activities were
measured at days 4, 7, and 14 postinfection on a subset of the cell
passages. -Gal activities detected at days 7 and 14 are expressed as
percentages of the activity detected at day 4, which is arbitrarily
considered to be 100%. (B) Hygr clones derived from
Bac-ITR/RepA- or Bac-ITR-infected 293 cells. From the experiment
described in panel A, cell aliquots were collected at day 4 postinfection and plated in hygromycin-selective medium. At 14 days
postinfection the clones were fixed and stained with 10% Giemsa and
counted under a microscope. Only clones containing more than 50 cells
were scored.
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To assess the influence of the AAV components on the frequency of
integration events, a sample of cells infected with either
Bac-ITR or
Bac-ITR/RepA was collected at day 4 postinfection and
plated
in hygromycin selection medium. As shown in Fig.
5B, infection
of
strain 293 cells with Bac-ITR/RepA resulted in a significantly
higher number of stable cell clones than did infection of cells
with
Bac-ITR. The increase in the number of Hyg
r clones ranged
in several experiments from 10- to 50-fold (data
not shown).
Five hundred Hyg
r clones were obtained by plating 20,000 cells infected with Bac-ITR/RepA. Based on the cloning efficiency
of
the 293 cells (50%) and on the percentage of the cells transduced
by
Bac-ITR/RepA (50%), we estimate that approximately 10% of the
transduced cells carrying the Hyg
r gene integrated into the
host genome. In addition, the majority
of the Hyg
r clones
(>90%) expressed
-Gal genes (as determined by histochemical
staining of the infected cells), suggesting that the entire ITR-flanked
transgene cassette had been inserted into the host chromosome.
These
results indicate that under both selective and nonselective
conditions,
baculovirus delivery of the
rep gene allows a very
efficient
transduction of ITR-flanked cassette, probably by mediating
its
insertion into the human genome.
Site-specific integration of ITR-flanked DNA.
It has been
shown that the rep gene mediates site-specific integration
of ITR-flanked DNA fragment into the aavs1 site (5, 37, 46).
To verify the efficiency of site-specific integration mediated by the
Bac-AAV vectors, Hygr clones from strain 293 cells infected
with Bac-ITR/RepA and Bac-ITR were isolated and expanded and total
genomic DNA was then subjected to Southern blot analysis. The
rearrangement of the aavs1 site resulting from the integration of the
ITR-flanked transgene cassette was assessed by using aavs1- and
Hyg--Gal-specific probes. Site-specific integration of the
transduced transgene cassette was scored when the aavs1-specific probe
recognized additional bands that were absent in mock-infected cells and
when the same additional bands were also detected by the transgene
probe.
The analysis of some of the strain 293 clones derived from infection
with Bac-ITR is shown in Fig.
6. The
genomic DNA extracted
from nine independent clones was digested
with
ApaI and analyzed
by Southern blotting.
ApaI was chosen because it cleaves once
in the aavs1
sequence but does not cleave within the transgene.
Thus, the insertion
of the transduced DNA into the aavs1 site
should be easily detectable
by the altered pattern of one of the
aavs1-derived restriction
fragments. The aavs1-specific probe
recognizes two bands of
approximately 2.5 and 2.8 kb (Fig.
6A,
lanes 2 to 10). Additionally,
the hybridization pattern of the
infected clone DNA is identical to
that of the mock-infected cell
DNA (Fig.
6A, lane 1). Single or double
bands ranging in size
from 8 to 20 kb were detected in selected DNA
clones when the
same genomic blot was hybridized with a probe specific
for the
transgene cassette. Thus, in all of the clones analyzed no
obvious
rearrangement of the aavs1 site could be ascribed to the
infection
of Bac-ITR, indicating that the integration of the transgene
occurred
at sites other than aavs1.
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|
FIG. 6.
Southern blot analysis of strain 293 Hygr
clones derived from Bac-ITR infection. Ten micrograms of genomic DNA
from each clone was digested with ApaI, fractionated on 1%
agarose gel, and transferred to a nylon membrane. (A) Hybridization to
an aavs1 probe. The two detected bands of roughly 2.5 and 2.8 kb
correspond to the aavs1 preintegration site. (B) Same membrane after
rehybridization to a transgene-specific probe. The positions of
molecular size standards (in kilobases) are indicated.
|
|
Figure
7 shows the hybridization pattern
obtained with the clones derived from the Bac-ITR/RepA-infected cells.
In 13 of 22
clones (59%) (Fig.
7A, clones C1, D1, B1, H1, G4, E1, A2,
H3,
F3, H4, C3, D2, and D4), rearrangement of the aavs1 region was
apparent. The same genomic blot was then hybridized with a transgene
probe to ascertain the presence of the transgene sequences within
the
rearranged aavs1 bands. In 8 of 13 clones (61%) (Fig.
7B,
clones C1,
B1, H1, G4, E1, C3, D2, and D4), bands were detected
which hybridized
to the transgene probe matching those obtained
with aavs1 probe (shown
with an arrow), indicating that the transgene
was indeed inserted in
the aavs1 site. Thus, site-specific integration
occurred in 8 of 22 clones (36%) analyzed. Additional transgene
bands detected only with
the transgene probe were present in half
the clones analyzed. These
bands might be due to rearrangements
in the aavs1 region flanking the
transgene sequences (
40) or
to multiple insertions at sites
other than aavs1.
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|
FIG. 7.
Southern blot analysis of strain 293 Hygr
clones derived from Bac-ITR/RepA infection. Conditions of infection and
DNA analysis were as described in Fig. 6. (A) Hybridization to aavs1
probe. (B) Same membrane hybridized to transgene probe. The upshifted
bands that are annealed to both probes are considered to be indicative
of site-specific integration and are indicated with arrows. The
positions of the molecular size standards (in kilobases) are
indicated.
|
|
To confirm the site-specific integration of the transduced ITR-DNA
cassette and to differentiate between single and multiple
integration
events into different sites, FISH analysis of metaphase
spreads was
performed on infected strain 293 cells by using the
aavs1- and
transgene-specific probes. Metaphases were scored as
positive only if
both probes were colocalized on both sister chromatids
of a given
chromosome.
At 10 days after infection with Bac-ITR/RepA, 400 Hyg
r
clones were pooled and analyzed by FISH. The transgene probe
colocalized
with the aavs1 probe in 10 of the 24 (41%) metaphases
analyzed
(Fig.
8B), whereas in the
remaining metaphases the transgene probe
was located on different
chromosomes that were not detected by
the aavs1 probe (Fig.
8A). The
aavs1 probe annealed to three or
four chromosomes 19 in most of
metaphases analyzed, in agreement
with the polyploid nature of this
cell line. No hybridization
of the transgene probe was detected on
mock-infected cells (data
not shown). Although FISH analysis is not
strictly quantitative,
the site-specific integration frequency of 41%
established by
FISH analysis of pooled cell clones is in good agreement
with
the integration frequency of 36% derived from Southern blot
analysis
of single clones. The colocalization of the transgene and
aavs1
probes was also observed upon FISH analysis of metaphases derived
from the individual clones that had been previously characterized
by
Southern blotting (data not shown).
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|
FIG. 8.
In situ hybridization of metaphase chromosomes from a
pool of 400 strain 293 Hygr clones derived from
Bac-ITR/RepA infection. Chromosome preparations were hybridized with a
Hyg--Gal-specific (in red) or aavs1-specific (in yellow) probe as
described in Materials and Methods. (A) The transgene is integrated
into a chromosome other than chromosome 19. (B) Colocalization of the
transgene and of the aavs1 probe.
|
|
Analysis of ITR-aavs1 junctions.
To precisely identify the
integration site of the transgene cassette, 293 and MRC-5 cells were
infected with Bac-ITR/RepA and the genomic DNA was extracted and
subjected to amplification of the ITR-aavs1 junction. For this purpose,
two sets of nested primers specific for the AAV terminal repeat and
chromosome 19 were used (17, 37). As a control, genomic DNA
from cells infected with Bac-ITR was also analyzed. As shown in Fig.
9A, specific DNA bands were amplified
from MRC-5 cells infected with Bac-ITR/RepA at MOIs of 100 and 500. The
size difference of the DNA bands probably reflects the different
junction species amplified from the population of transduced cells. In
contrast, no specific product was detected with mock- or
Bac-ITR-infected cells. A similar protocol was utilized to amplify the
ITR-aavs1 junction from Bac-ITR/RepA-infected 293 cells. The amplified
DNA bands were cloned and sequenced, and the sequence data is shown in
Fig. 9B. In MRC-5 cells, the insertion of the ITR-flanked transgene
cassette was mapped at nucleotide 1111 of aavs1, where a short homology
between the AAV ITR and aavs1 can be identified in the GCC triplet. A
deletion of 43 and 83 bases within the ITR sequence was also
identified. Similarly, the ITR-aavs1 junctions amplified from pooled
Hygr 293 clones indicated that the insertion of the
transgene cassette had occurred at nucleotides 1012, 1080, and 1096 of
aavs1. Interestingly, in clone 293-2 an insertion of three nucleotides
was detected at the junction between the ITR and aavs1. A larger insert
of 101 nucleotides was identified in clone 293-1. This insert is partially homologous to sequence X62488 deposited in the GenBank database by Samulsky and coworkers and is derived from amplification of
the ITR-aavs1 junction from AAV-infected cells (40). Taken together, these results indicate that, analogous to what has been observed following AAV infection and in transfection of AAV-derived plasmids (5, 37, 42, 46), baculovirus-mediated transduction of human cells results in the integration of the ITR-flanked DNA cassette in a specific region of the aavs1 site.
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FIG. 9.
(A) PCR amplification of the ITR-aavs1 junction in
baculovirus-infected strain 293 cells. Cells were infected at the
indicated MOI and were collected 4 days postinfection; total DNA was
prepared and subjected to nested PCR amplification as described in
Materials and Methods. Bands corresponding to primers and amplified
junctions are indicated. (B) ITR-aavs1 junction sequences from
Bac-ITR/RepA-infected 293 and MRC-5 cells. Common bases at the
ITR-aavs1 junction are underlined. Sequences not belonging to ITR or
aavs1 are in boldface. X62488 was identified in GenBank with a Blast
search. Sequences are depicted in ITR, aavs1 order.
|
|
| |
DISCUSSION |
In this study, we described the construction of a novel hybrid
virus which combines the remarkable transducing efficiency of
baculovirus with the unique property of site-specific integration characteristic of AAV. The Bac-AAV vector constructed displays the
following properties: genome stability upon virus amplification, highly
efficient transduction of primary cells, and targeted integration of
the ITR-flanked DNA. Thus, this hybrid virus is ideal for the delivery
of large DNA segments to both culture and primary cell lines where the
gene of interest flanked by the AAV terminal repeats can be maintained
and expressed for an extended period of time.
The strategy implemented in the construction of the hybrid Bac-AAV
vector is based on the observation that baculovirus readily infects
hepatocytes without any apparent toxic effect on the transduced cells
(8, 19). Interestingly, baculovirus can infect not only
hepatic cells, but also other cell types such as the human fibroblast
cell strain MRC-5 (Fig. 2), human embryonic kidney cells strain 293 (Fig. 3), HeLa cells (54), and primary rat fibroblasts (data
not shown). The efficiency of baculovirus transduction of these cell
types is quite high and reaches levels comparable to those observed
with hepatoma cell line Huh-7 (data not shown). Thus, although the
mechanism of viral uptake is not clear, these results indicate that the
ability of baculovirus to express a reporter gene is quite general and
can be observed in a variety of cell types.
Rep polypeptides are expressed in Sf9 cells (Fig. 4), indicating that
the p5 and p19 promoters that govern transcription of the
rep gene are also functional in insect cells. Rep proteins inhibit the replication of a number of viruses, including SV40 (6), human immunodeficiency type 1 (2), bovine
papillomavirus type 1 (18), and Ad (51).
Apparently, rep expression does not interfere with
baculovirus replication since hybrid Bac-AAV viruses carrying the
rep gene grow to titers comparable to those of other
recombinant baculoviruses (data not shown). Interestingly, hybrid
vectors carrying both the rep gene and the ITR-flanked transgene cassette can be amplified only where the rep gene
is positioned in an antisense orientation with respect to the pPolh promoter (Fig. 1 and 4). The reduction in Rep protein expression in
cells infected with Bac-ITR/RepA may be explained, at least in
part, by the transcription of antisense Rep RNA mediated by the pPolh promoter, which is known to be very active in these cells
(36). The mechanism by which the expression of Rep
polypeptides may compromize the stability of the Bac-AAV genome is not
clear, but it can be assumed that the excision of the ITR-flanked
transgene cassette is mediated by the expression of Rep polypeptides
upon baculovirus amplification in Sf9 cells. Although AAV replication requires Ad helper functions (7), it has been demonstrated by transfection studies of AAV plasmids that overexpressed Rep proteins
can excise the ITR-flanked DNA, converting it into monomer-sized replicative intermediate molecules even in the absence of Ad functions (32). Thus, although we have not assessed the functionality of Rep polypeptides in insect cells, a similar event may have occurred
in Sf9 cells infected with the Bac-ITR/RepS, leading to disruption of
the Bac-AAV genome structure. The stability of the Bac-ITR/RepA may in
turn be explained by the reduced amount of Rep polypeptides which, in
the context of the Sf9 cells, may not have been sufficient to mediate
the excision of the ITR-flanked cassette.
The correlation between the level of rep gene expression and
the loss of the ITR-flanked DNA has important implications for the
construction of mammalian hybrid vectors carrying both the rep gene and the ITR-flanked DNA. If these recombinant
vectors are amplified in mammalian cells, the efficiency of
rep-mediated excision of the ITR cassette may jeopardize the
integrity of the genome structure of the recombinant virus. Thus, the
construction of chimeric vectors carrying these genetic elements may
require a tight control of rep gene expression.
Interestingly, the construction of a hybrid herpesvirus-AAV vector was
recently reported (23). This vector prolongs expression of
the -Gal reporter gene, albeit only for a few days. The overall
genomic structure of the hybrid virus was not described, and thus it is
entirely possible that, analogous to our observations with
Bac-ITR/RepS, the expression of the Rep polypeptides may have caused
partial loss of the ITR-flanked cassette.
The prolonged -Gal expression in the presence of the rep
gene (Fig. 5) reflects an increase in integration levels stimulated by
the Rep polypeptides, demonstrating the validity of the Bac-AAV vector
in ensuring prolonged persistence of the transgene cassette. This
conclusion is also supported by the observation that the number of
Hygr strain 293 clones obtained with Bac-ITR/RepA infection
is 10- to 50-fold higher than with Bac-ITR alone (Fig. 5). A similar observation based on transfection of 293 cells with plasmids carrying the rep gene has been recently reported (5, 46).
We do not know if the higher transduction efficiency based on the
expression of rep is restricted to strain 293 cells and may
be attributed to the E1A-mediated induction of the p5 promoter or if
the enhanced integration is observable in other cell types. To this
end, we are currently examining the integration efficiency of Bac-AAV in other cell types.
The fate of the ITR-flanked transgene in selected clones was assessed
by Southern blot analysis, FISH, and PCR amplification of the ITR-aavs1
junction. In agreement with several reports (5, 37, 37a,
46), targeting of the ITR-flanked DNA to aavs1 is dependent on
the expression of the rep gene since no integration of the
ITR-transgene in chromosome 19 was detected upon infection of the 293 cells with Bac-ITR (Fig. 6). In contrast, Bac-ITR/RepA infection of 293 cells results in a 36 to 41% frequency of site-specific integration
(Fig. 7 and 8). The frequency of integration observed upon baculovirus
infection of 293 cells is comparable to that detected in these cells
upon infection with recombinant AAV virus or with the transfection of
AAV-derived plasmids (5, 37, 42, 46). However, in a study of
bronchial epithelial cells, in 94% of the cells containing integrated
wild-type AAV, the virus genome was mapped on chromosome 19 by FISH
(25). Additionally, approximately 70% of latently infected
cell lines that were analyzed in another survey were found to contain
AAV inserted into aavs1 (42). Thus, the integration
efficiency detected in other cell types with wild-type AAV appears to
be significantly greater than that observed in 293 cells upon either
plasmid transfection or Bac-AAV infection. The biological significance
of this difference in integration efficiency is not clear; however, it
is tempting to speculate that it may reflect differences in the
concentration of specific host factors that may contribute to targeting
to the aavs1 site of the AAV-derived DNA.
Southern blot analysis of the selected strain 293 clones revealed the
presence of several bands corresponding to rearranged aavs1 sequences
that can be accounted for by the insertion of the transgene cassette
(Fig. 7). Also, several bands hybridized only by the aavs1 probe
indicate that rearrangement of the aavs1 as a consequence of
rep expression may have taken place, much as has been
observed upon transfection of AAV-derived plasmids and subsequent
selection of neomycin-resistant 293 clones (5). Interestingly, in half of the clones analyzed the probe specific for
the -Gal-Hygr genes detected an additional band which
varied in size. An additional band was also detected in clones infected
with Bac-ITR. Although this band appears less intense, it probably
represents a second integration event. In line with this hypothesis,
FISH analysis revealed the presence of second integration events in a
minority of the metaphases analyzed (Fig. 8).
The 6.5-kb transgene cassette inserted between the ITRs contains the
Hygr gene and the -Gal gene that were coselected in
the presence of hygromycin in almost all of the clones analyzed (data
not shown). Although this cassette is 2 kb longer than the
wild-type virus genome, site-specific integration of the ITR-flanked
DNA was observed when the rep gene was carried on the same
virus (Fig. 7 and 8). We have not examined the fine structure of the
ITR-flanked DNA inserted into the aavs1 site. However, the size of the
bands hybridized by both the transgene and aavs1 probes is consistent
in most clones with insertion of the full-length ITR-flanked transgene
cassette. This possibility is also supported by the observation that
both reporter genes are expressed in all clones and, most
importantly, in those clones where only one upshifted band is
detected. It is, however, possible that fine rearrangements at the
integration sites have taken place, disrupting the structure of the
ITR-flanked DNA without interfering with the expression of both genes.
The observation that rep-mediated site-specific integration
in 293 cells is accompanied by rearrangements of the integration site
and, possibly, of the inserted transgene cassette is not unprecedented
(5). Thus, it is not surprising that rearrangement of the
aavs1 region is detected upon infection of 293 cells with Bac-ITR/RepA
and subsequent selection of Hygr clones. Although the
biological reasons for such rearrangements are not clear, it is
important to point out that such genome rearrangements have been mostly
documented with established cell lines. It would be of interest to
determine whether the same rearrangements are detected in primary
cultures. Nonetheless, this result indicates that integration is not
limited by the size of the DNA flanked by the AAV terminal repeats and
can occur with DNA segments larger than the wild-type AAV genome.
Important large genes (such as the dystrophin gene) can, at least in
theory, be transduced and integrated by using this system.
rep-mediated integration also occurs in the human fibroblast
cell strain MRC-5 after Bac-ITR/RepA infection. This conclusion is
based on the PCR amplification of the ITR-aavs1 junction that localizes
the insertion of the ITR-flanked DNA cassette in the same region of
aavs1 as described previously for AAV (40) (Fig. 9). Thus,
although no further work has been carried out to characterize the
structure of the inserted DNA or to determine the efficiency of
site-specific integration, this result extends the use of the hybrid
Bac-AAV virus to human diploid cells. The ITR-aavs1 junctions of the
infected MRC-5 cells show similarities to those generated by wild-type
AAV. In fact, as previously reported (40), several nucleotides are found in common between the ITR and aavs1 at the viral-cellular junction (Fig. 9). In contrast, the insertion of a short
stretch of nucleotides in between the ITR-cellular junction was
detected in two junctions derived from strain 293 infected cells. The
mechanism by which these insertions have been generated is not clear,
but it is unlikely that they are artifacts of PCR amplification
since the longer insertion shows 90-nucleotide sequence identity
to a similar sequence reported by Samulsky and coworkers (40). We have observed similar insertions in Huh-7 cells
infected with wild-type AAV (37). Although the
biological significance of these insertions is not clear, it may be
related to differences between the diploid cell strain MRC-5 and the
polyploid cell lines Huh-7 and 293.
The results presented in this work indicate that transgene excision
from a double-stranded DNA baculovirus backbone vector and subsequent
integration into the human genome occur with significant frequency.
Thus, this study can be considered an important step towards improving
the use of baculovirus as a vector for gene transfer since it
demonstrates the feasibility of using hybrid Bac-AAV virus for in vitro
gene transfer in human cells. Baculovirus infection in vivo,
like retrovirus infection, is hampered by serum components, namely, by
complement factors (41). Therefore, the use of this system
may be limited to ex vivo gene delivery. However, the data presented
here indicate that baculovirus infection and permanent transduction of
the target cells is not limited to hepatic cells. The demonstration
that hybrid viral vectors carrying specific genetic elements of the AAV
virus can direct integration of a selected DNA sequence is an important
starting point for the construction of chimeric viral vectors suitable
for in vivo gene delivery.
| |
ACKNOWLEDGMENTS |
We thank A. Sgura and D. Cimini for in situ hybridization
analysis of infected cells and E. Fattori, A. Nicosia, and C. Toniatti for critical review and helpful suggestions. We also thank J. Clench
for editorial assistance, M. Emili for graphics, and P. Neuner for
oligonucleotide synthesis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IRBM P. Angeletti, Via Pontina Km 30,600, 00040 Pomezia, Italy. Phone:
39-6-91093-443. Fax: 39-6-91093-225. E-mail:
lamonica{at}irbm.it.
| |
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Abstract
Introduction
