Conditional Site-Specific Integration into Human Chromosome 19 by Using a Ligand-Dependent Chimeric Adeno-Associated Virus/Rep Protein

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Conditional Site-Specific Integration into Human Chromosome 19 by

Using a Ligand-Dependent Chimeric Adeno-Associated

Virus/Rep Protein

DANIELA RINAUDO, STEFANIA LAMARTINA, GIUSEPPE ROSCILLI, GENNARO CILIBERTO,

CARLO TONIATTI*

Department of Genetics, Istituto di Ricerche di Biologia Molecolare, I.R.B.M.—Piero Angeletti,

00040 Pomezia (Rome), Italy

Received 7 May 1999/Accepted 21 September 1999

It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into

a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian

viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The

inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78

and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is

also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or

have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form

of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain

of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist

RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various

assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a

ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement

at AAVS1.

One of the major goals of gene therapy is to develop safe

and reliable systems for the prolonged expression of a

thera-peutic gene (9). This result can be achieved by promoting

integration of the desired DNA sequence into a predetermined

site on the host genome (14). Therefore, considerable interest

has been raised by the observation that the adeno-associated

virus (AAV) integration machinery can be used for directing

the integration of a transgene into a specific site on the human

genome (31, 44).

AAV is a human defective parvovirus whose single-stranded

genome, 4.7 kb long, is flanked by two inverted terminal

re-peats (ITRs) and comprises two open reading frames (ORFs)

called

rep

and

cap

, which code for nonstructural and structural

proteins, respectively (4). AAV replicates only in cells

coin-fected by a helper virus, such as adenovirus (Ad) or herpes

simplex virus, or undergoing genotoxic stress, such as UV

treatment or X-ray irradiation (4). Under conditions which are

not permissive for replication, AAV establishes a latent

infec-tion in which the viral genome integrates stably and efficiently

into a defined region, AAVS1, of human chromosome 19

(q13.3-qter) (4, 26, 27, 44, 46).

The precise molecular mechanisms underlying AAV

inte-gration have not yet been fully elucidated, but it has been

clearly established that two viral elements are required: the

145-bp ITRs and the Rep78 and/or Rep68 protein encoded by

the

rep

ORF (14, 24, 31, 65). Rep78 and Rep68, which are

respectively 623 and 536 amino acids (aa) long, are expressed

from alternatively spliced transcripts initiated at the same

pro-moter (the p5 propro-moter) and therefore differ only at their

carboxy termini (4). The two proteins, which are essential not

only for integration but also for AAV replication, have several

biochemical activities in common: they both interact with a

specific DNA sequence (Rep binding site) and they both have

a strand- and site-specific endonuclease activity and an

ATP-dependent DNA-DNA and DNA-RNA helicase activity (22,

23). In addition, they are able to modulate the activity of

endogenous as well as heterologous promoters (21, 29, 39). In

vitro and ex vivo experiments suggest that the earliest step in

the integration process is when Rep78 and/or Rep68 tethers

the two Rep binding sites present in the AAV ITR and in

AAVS1 (8, 9, 15, 60). Following the formation of this

Rep-mediated complex between AAV DNA and its target site in

chromosome 19, Rep78 and/or Rep68 is postulated to

specif-ically nick DNA within the ITR and AAVS1; subsequently,

integration is likely to occur via a nonhomologous

recombina-tion process mediated by replicarecombina-tion of the integrating DNA

with the active participation of host factors responsible for

DNA synthesis (32, 33, 56, 65).

The limited length of the DNA sequence which can be

packaged into AAV particles, coupled with the need to

main-tain the

rep

ORF, precludes the use of AAV itself as a delivery

vector for promoting site-specific integration of a transgene

(14). However, recent results indicate that the AAV

integra-tion machinery works quite efficiently also when incorporated

into a variety of alternative nonviral and viral delivery systems

(31). It has in fact been demonstrated that Rep78 and/or

Rep68, when delivered to cells either as an expression plasmid

or as a recombinant protein, promotes the site-specific

inte-gration of an AAV ITR-flanked cassette contained in the same

or in a separate plasmid (2, 30, 47, 52, 55). Furthermore, it was

recently shown that a baculovirus/AAV hybrid vector, which

carries both an AAV ITR-flanked transgene and a Rep

ex-pression cassette, was capable of driving integration of the

ITR-flanked transgene at AAVS1 (38).

* Corresponding author. Mailing address: Istituto di Ricerche di

Biologia Molecolare, I.R.B.M.—P. Angeletti, Via Pontina Km 30.600,

00040 Pomezia (Rome), Italy. Phone: 91093668. Fax:

39-06-91093654. E-mail: [email protected].

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In considering the Rep78/68-based integration system as a

new approach to gene therapy, it would be highly desirable to

restrict Rep78/68 activity in target cells only to the time

re-quired for site-specific integration to occur, in order to

mini-mize additional and possible detrimental effects. In fact, it has

been shown that Rep proteins down-regulate the expression of

human genes such as c-H-

ras

, c-

fos

, c-

myc

, and c-

sis

(18, 19, 62)

and can inhibit the proliferation of some cell lines (4, 68). A

further fact for consideration is that Rep-mediated integration

at AAVS1 can lead to nonspecific genomic rearrangements at

the same locus, and the frequency and severity of these might

be attenuated or suppressed by setting a time limit to the

activity of Rep proteins in target cells (2, 52).

With this in mind, we decided to generate an inducible form

of Rep78/68 whose activity could be controlled by an externally

added small-molecule ligand. In this paper we describe the

construction of a ligand-dependent Rep chimeric protein,

made up of a C-terminal Rep68 deletion mutant fused with a

truncated form of the hormone binding domain (HBD) of the

human progesterone receptor (PR), known to interact with the

synthetic steroid RU486 but not with endogenous

progester-one (3, 5, 57). This Rep/HBD fusion protein displays strictly

RU486-dependent activity in a wide array of functional assays

and promotes site-specific integration at AAVS1 without

ma-jor nonspecific genomic rearrangements.

MATERIALS AND METHODS

Plasmid construction and site-directed mutagenesis.Expression vectors for Rep78 and Rep68 (plasmids pCMV/Rep78 and pCMV/Rep68) were obtained by cloning the coding regions for Rep78 and Rep68 under the control of the cytomegalovirus (CMV) enhancer-promoter element contained in plasmid pcD-NAIII (30). To obtain the cDNAs coding only for Rep78 or Rep68, therepORF, spanning from nucleotides 321 to 2252 of the AAV-2 genome (51), was subjected to PCR-based mutagenesis with the AAV-2 genome contained in plasmid pTAV2 (17) as a substrate. To generate the cDNA coding for Rep78, the internal start methionine for the small Rep proteins (Rep52 and Rep40) was mutated to glycine (nucleotides 993 to 995, ATG changed to GGA) and the splice donor site required for the expression of the spliced version Rep68 was eliminated by introducing a G-to-A transversion at nucleotide 1907 (30). The cDNA for Rep68 was obtained by first mutating the internal translational AUG as described above and then deleting the entire intron (positions 1907 to 2227) (30). C-terminal Rep68 deletion mutants (Rep1–484, Rep1–491, Rep1–502, and Rep1–520) were obtained by inserting stop codons at appropriate positions in the context of pCMV/Rep68 by PCR-mediated site-specific mutagenesis (1). Expression plas-mid pCMV/Rep contains the wholerepORF, with no mutations, cloned down-stream of the CMV enhancer/promoter in plasmid pcDNAIII and codes for all four species of Rep. The cDNAs coding for the C-terminal deletion mutants were then cloned into pcDNAIII, thus creating expression plasmids pCMV/ Rep1–484, pCMV/Rep1–491, pCMV/Rep1–502 and pCMV/Rep1–520. All Rep/PR fusions (Rep78/PR, Rep78int/PR, PR/Rep78, Rep68/PR, Rep68int/PR, PR/Rep68, Rep1–491/P, and Rep1–484/Pn) were generated by a PCR-based mutagenesis strategy: their corresponding cDNAs were cloned into pcDNAIII downstream of the CMV enhancer/promoter element, thus generating the ex-pression vectors pCMV/Rep78/PR, pCMV/Rep78int/PR, pCMV/PR/Rep78, pCMV/Rep68/PR, pCMV/Rep68int/PR, pCMV/PR/Rep68, pCMV/Rep1– 491/P, and pCMV/Rep1–484/Pn. The sequences of all mutants and fusions were verified by the dideoxy-sequencing method (1). The sequences of all oligonucle-otides used for PCRs are available on request. Plasmid p5/LUC was constructed as follows: the p5 promoter region (nucleotides 1 to 319 of the AAV-2 sequence) (51) was PCR amplified from psub201 and then cloned as anEcoRV-HindIII fragment upstream of thelucgene contained in plasmid pGL2-Basic (Promega). Plasmid ITR/Hook-Neo, containing the expression cassette for the neomycin resistance gene (neo) and for the Hook gene, has been described previously (30). Plasmid pT7bhPRB-891, containing the C-terminal deletion of the HBD of the human progesterone receptor, was a generous gift of B. O’Malley and S. Tsai.

Cell culture and transfections.293, HeLa, and Hep3B cells were propagated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum plus glutamine and antibiotics at 37°C in 5% CO2. All transfections were

per-formed by the calcium phosphate procedure (1).

Immunoblot analysis of transiently transfected cells and of stable transfor-mants.For analysis of Rep expression, 3⫻105_{cells (Hela, 293, or Hep3B) were}

transfected with 10␮g of the various expression plasmids. At 48 h after trans-fection, the cells were washed with phosphate-buffered saline (PBS) and total cellular extracts were prepared as described previously with minor modifications (53). Briefly, the cells were lysed in 10 mM Tris-HCl (pH 8.0)–5 mM EDTA–1%

sodium dodecyl sulfate (SDS) by passage through a 1-ml syringe. The lysate was precipitated with 10% trichloroacetic acid at room temperature for 15 min and then left in ice for additional 10 min. After centrifugation at 12,000⫻gfor 15 min, pellets were washed in ice-cold acetone, resuspended in 60␮l of sample buffer, and incubated at 65°C for 15 min and then at 100°C for 3 min. Equivalent amounts of proteins were fractionated on an 8% polyacrylamide–SDS gel, trans-ferred to nitrocellulose filters, and detected by sequential incubation with a rabbit polyclonal antibody directed against AAV Rep proteins (dilution, 1:1,000) and then with an alkaline phosphatase-conjugated rat polyclonal anti-rabbit immunoglobulin G (IgG) antiserum (Promega no. S3731; dilution, 1:4,000). The polyclonal antiserum against Rep proteins was obtained by immunizing rabbits with purified recombinant Rep68 produced inEscherichia coli(30) and recog-nizes all four species of Rep.

To check the expression of the Hook gene product (sFv/PDGFR fusion pro-tein) (6) in the stable transfectants, total cellular extracts were prepared as described above, fractionated on an SDS–12% polyacrylamide gel, and trans-ferred to nitrocellulose membranes. To detect the sFv/PDGFR fusion protein, the membranes were incubated first with monoclonal antibody 9E10.2 (dilution, 1:500), which recognizes the Myc.1 epitope tag (13) present as a tandem repeat near the transmembrane domain of sFv/PDGFR (6), and then with an alkaline phosphatase-conjugated goat polyclonal anti-mouse IgG antiserum (Sigma no. A7434; dilution, 1:2,000). In all immunoblotting experiments, 5% nonfat dry milk in TBST (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) was used as a blocking agent and for diluting the various antibodies.

Immunofluorescence experiments. For immunofluorescence assays, cells (Hep3B, 293, or HeLa) were grown on glass coverslips and transfected with 10

␮g of the expression vectors for the various Rep derivatives. At 36 h posttrans-fection, the cells were fixed with 3% formaldehyde in PBS at room temperature for 20 min, washed with PBS, and incubated in 0.1 M glycine in PBS at room temperature for 10 min. Subsequently, the cells were permeabilized by incuba-tion at room temperature for 5 min with 0.1% Triton X-100 in PBS, and after a washing step, they were incubated for 20 min with the rabbit polyclonal antibody directed against AAV Rep proteins (diluted 1:200 in 10% goat serum in PBS). They were then washed with PBS and incubated for an additional 20 min with a fluorescein-conjugated goat anti-rabbit IgG (Pierce No. 31572; diluted 1:100 in 10% goat serum in PBS). After sequential washing steps in PBS and distilled water, the coverslips were mounted in Moviol containing 1 mg of para -phe-nylenediamine per ml and photographed by epifluorescence on a Leica Diaplan photomicroscope with fluorescein filters and a 63x planar objective.

p5 promoter repression assay.For analysis of Rep repressing activity, 3⫻105

293 or HeLa cells were seeded in a 60-mm-diameter dish; 20 h later, they were cotransfected with 50 ng of the various expression plasmids and 5␮g of p5/LUC. At 15 h later, the cells were washed, and 100 nM RU486 was added to some of the cells. After 36 h, the cells were collected in 40 mM Tris–1 mM EDTA–150 mM NaCl (pH 7.5), centrifuged, resuspended in 250 mM Tris (pH 8.0), and lysed by three freeze-thaw cycles. Cell debris was pelleted by centrifugation, and the supernatant was used for quantitation of luciferase activity as described previ-ously (1) by using a Lumat luminometer (Berthold). Luciferase activity in cell lysates was normalized to the corresponding protein concentration.

Rescue-replication assay.A total of 1.2⫻106_{HeLa, 293, or Hep3B cells were}

seeded in a 10-mm-diameter dish and 18 h later were infected with Ad2 at a multiplicity of infection of 10. After 2 h of incubation, the medium was changed and the cells were transfected with 10␮g of plasmid ITR/Hook-Neo with or without 10␮g of the expression vectors for wild-type wt Rep78, Rep68, or their various derivatives. At 15 h after transfection, the cells were washed and incu-bated with fresh medium containing or not containing 100 nM RU486. At 48 h later, low-MrDNA samples were isolated from cells by the procedure described

by Hirt (20), digested extensively withDpnI (62), and analyzed by Southern blotting with a32_{P-labeled DNA probe specific forneosequence (1, 30).}

In vitro translations.Rep68, Rep1–484, Rep1–491, Rep1–502, and Rep1–520 were translated in vitro from plasmids pCMV/Rep68, pCMV/Rep1–484, pcDNA/Rep1–491, pcDNA/Rep1–502, and pcDNA/Rep1–520, respectively, with the TnT-T7 coupled reticulocyte lysate system (Promega) as recommended by the manufacturer. The quantity of proteins used for in vitro experiments was normalized by densitometric analysis (with the GS-700 imaging densitometer with Molecular Analyst software [Bio-Rad]) of SDS-polyacrylamide gels loaded with increasing quantities of the various in vitro translation products.

Electrophoretic mobility shift assays.Electrophoretic mobility shift assays were performed with 20,000 cpm of32_{P-end-labeled AAV ITR. Reaction}

mix-tures (10␮l) contained 10 mM HEPES-NaOH (pH 7.9), 8 mM MgCl2, 40 mM

KCl, 0.2 mM dithiothreitol, 1␮g of poly(dI-dC), and different amounts of the in vitro-translated proteins. Following a 20-min incubation at room temperature, 2

␮l of 20% Ficoll was added, samples were loaded on 4% polyacrylamide gels (acrylamide/bisacrylamide ratio, 29:1; 0.5⫻Tris-borate-EDTA [TBE]) and elec-trophoresed at room temperature at 10 V/cm. The gels were dried and subjected to autoradiography for 6 h at⫺80°C.

trsendonuclease assay.Endonuclease assays were performed essentially as previously described, using substrates with a single-stranded terminal resolution site (30, 48). Briefly, the double-strandedXbaI-PvuII fragments from plasmid psub201 (45) containing the AAV ITRs, were dephosphorylated with calf intes-tinal alkaline phosphatase, purified from agarose gels, 5⬘-end labelled with polynucleotide kinase, and loaded on 6% polyacrylamide sequencing gels. The

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plus (trs⫹_{) strand was eluted from the gels in 0.5 M ammonium acetate (pH}

8.0)–1 mM EDTA and annealed. For the endonuclease assay, the reaction mixture (20␮l) contained 25 mM HEPES-KOH (pH 7.5), 5 mM MgCl2, 0.2 mM

EGTA, 1 mM dithiothreitol, 0.4 mM ATP, 0.2␮g of bovine serum albumin, 1␮g of poly(dI-dC), 20,000 cpm of32_{P-end-labeled substrate, and different amounts}

of the in vitro-translated proteins. The reaction mixtures were incubated for 1 h at 37°C, the reactions were stopped with proteinase K for 30 min at 65°C, and the products were subjected to phenol-chloroform extraction, ethanol precipitated, and analyzed on an 8% sequencing gel.

PCR assay for site-specific integration.For each assay, 1.2⫻106_{HeLa cells}

were seeded in a 10-mm-diameter dish and 20 h later were cotransfected with 10

␮g of the ITR/Hook-Neo plasmid alone or with 10␮g of one of the Rep or Rep/PR fusion expression plasmids. Transfected cells were serially passaged for 14 days. Total genomic DNA was then extracted (1), and 500 ng was used as a template for two consecutive rounds of nested PCR amplifications performed with two matched couples of ITR-specific and AAVS1-specific primers as de-scribed previously (30). The PCR products were resolved on a 1.5% agarose gel, blotted onto a nylon membrane, and hybridized with an AAVS1-specific probe spanning nucleotides 210 to 1207 of the published AAVS1 sequence (27). For molecular cloning of the amplified ITR/AAVS1 junctions, the products of the second round of amplification which were detectable on ethidium bromide-stained agarose gels were purified, filled with Klenow enzyme, cloned into plas-mid pBluescript II SK(⫹) (Stratagene), and sequenced by the dideoxy sequenc-ing method (1).

Isolation and Southern blot analysis of neomycin-resistant clones.A total of 106_{HeLa cells were seeded in a 10-mm-diameter dish and 20 h later were}

cotransfected with 10␮g of ITR/Hook-Neo plasmid and 10␮g of plasmid pCMV/Rep68 or pCMV/Rep1–491/P. At 15 h later, after a washing step, the cells cotransfected with pCMV/Rep1–491/P were treated for 12 h with 100 nM RU486 or left untreated. Subsequently, the medium was changed again and the cells incubated in normal medium for an additional 36 h. Selection was then carried out by growing cells in the presence of 700␮g of G418 per ml (70.6% active; effective concentration, 494.2␮g/ml). After 14 to 18 days of growth in selective medium, single-cell neomycin-resistant clones were isolated and ex-panded. For Southern blot analysis, genomic DNA was extracted and purified by standard procedures (1), digested with the restriction enzymeBamHI, and blot-ted onto a nylon membrane, which was sequentially hybridized with AAVS1- and neo-specific probes by published methods (1). The AAVS1-specific probe was a DNA fragment (derived from plasmid pRVK [K. Berns, Cornell Medical School, Ithaca, N.Y.]) spanning nucleotides 1 to 3525 of AAVS1, which was labelled with

32_{P by the random-priming reaction. A DNA fragment of 630 bp was used as a}

template in the random-priming reaction for generating theneo-specific probe.

RESULTS

RU486-dependent nuclear localization of full-length Rep78

and Rep68 fused to the HBD of the progesterone receptor.

To

obtain ligand-dependent Rep78 and/or Rep68 proteins, we

decided to generate fusions with a 42-aa C-terminal deletion of

the human PR891-HBD (57). PR891-HBD (aa 642 to 891 of

the human PR) was chosen because it binds synthetic

proges-terone antagonists, such as RU486, but not the progesproges-terone

or other natural steroids (5, 57); therefore, the activity of

heterologous proteins fused to PR891-HBD cannot be affected

by natural steroids (25, 50).

Since it is not possible to exactly anticipate on a rational

basis the position in which the HBD must be fused with the

heterologous moiety to obtain a stable, properly folded and

ligand-dependent chimera, we constructed several different

fu-sions. In four of them, the HBD was cloned either at the N

terminus (PR/Rep78 and PR/Rep68) or at the C terminus

(Rep78/PR and Rep68/PR) of both Rep78 and Rep68 (Fig.

1A). We also constructed two additional chimeras in which the

HBD was introduced at the level of the splicing site

(Rep78int/PR and Rep68int/PR [Fig. 1A]). In fact, evidence

reported in the literature suggests that the splicing site in the

rep

ORF delimits a distinct C-terminal domain of the protein

(12, 21); we therefore reasoned that insertion at this point

should not dramatically affect protein folding and, with respect

to C-terminal fusions, should bring the HBD in closer contact

with the regions of Rep78/68 known to be important for DNA

binding and nicking (11, 34, 37, 55, 61, 67).

Expression vectors for the six fusions were transfected into

human adenocarcinoma-derived HeLa cells, and their

expres-sion levels were assessed by Western blotting experiments. As

shown in Fig. 1B, the N-terminal fusions were undetectable in

transfected cells (Fig. 1B, lanes 1 and 4) while the expression

levels of the other four fusions were comparable to that of wt

Rep68 (Fig. 1B, lanes 2, 3, 5, and 6). Similar results were

obtained in Ad-transformed human embryonic kidney 293 cells

and human hepatoma Hep3B cells (not shown). Notably,

N-terminal fusions were produced by in vitro translation as

effi-ciently as the wt Rep proteins were (data not shown),

suggest-ing that their low expression in cells is due to intracellular

instability. Further analysis was therefore restricted only to the

four chimeric proteins expressed in vivo, namely, Rep78/PR,

Rep78int/PR, Rep68/PR, and Rep68int/PR.

Rep78 and Rep68 are intranuclear proteins (21, 66);

there-fore, before testing the functional activity of our four chimeric

proteins, we first analyzed their intracellular localization.

Ex-pression vectors for Rep78/PR, Rep78int/PR, Rep68/PR, and

Rep68int/PR were thus transfected into Hep3B cells treated or

not treated with 100 nM RU486, and the subcellular

distribu-tion of the fusion proteins was monitored by

immunofluores-cence analysis (see Materials and Methods). Control

experi-ments were performed with Rep78 and Rep68. For each type

of protein, at least 1,000 stained cells were analyzed and

clas-sified into three categories: N, containing cells showing

pre-dominantly nuclear staining; C, containing cells with

predom-inantly cytoplasmic staining; and N

⫽

C, containing cells in

which cytoplasm and nucleus are equally stained. The results

were expressed as the percentages of stained cells in each

category; they are summarized in Table 1, and examples of the

immunocytochemical presentation of cells scored in the three

different categories are shown in Fig. 1C. As expected, Rep78

and Rep68 showed a clear nuclear localization (N

⫽

95%

[Table 1 and Fig. 1C]). In contrast, Rep78/PR, Rep78int/PR,

Rep68/PR, and Rep68int/PR were confined predominantly to

the cytoplasm in the absence of RU486 (C

ⱖ

90% and N

ⱕ

2%

[Table 1]) but migrated into the nuclei following hormone

treatment (N

ⱖ

90% [Table 1]): representative results

ob-tained with the Rep68/PR fusion are shown in Fig. 1C. These

results were also obtained with HeLa and 293 cells (not shown)

and demonstrated that within the sensitivity limits of

immuno-fluorescence, nuclear translocation of the fusion proteins was

under quite stringent hormonal control.

The activity of full-length Rep78 and Rep68 fused to PR

HBD is not hormone dependent.

To determine whether the

activity of the four chimeric constructs was hormone

depen-dent, we tested them in a transcription repression assay. Rep78

and Rep68 inhibit transcription starting from the AAV p5

promoter (21, 29, 39). Expression vectors for the Rep/PR

fu-sions were thus cotransfected with a plasmid containing the

luciferase gene under the control of the p5 promoter (plasmid

p5/LUC), in the presence or absence of 100 nM RU486. 293

cells were selected as recipient cells, because the p5 promoter

is known to be highly active in this cell line (29). As expected,

cotransfection of p5/LUC with expression vectors for Rep78

and Rep68 (pCMV/Rep78 and pCMV/Rep68, respectively)

led to the complete inhibition of luciferase activity (98%

re-pression [Fig. 2A]). The four chimeric proteins Rep78/PR,

Rep78int/PR, Rep68/PR, and Rep68int/PR also acted as

strong repressors in the absence of hormone treatment (Fig.

2A). Similar results were observed in HeLa cells, in which the

p5 promoter had a lower but still detectable activity (reference

39 and data not shown).

The four chimeric proteins were further tested in a

rescue-replication assay commonly used to monitor the ability of

Rep78 and Rep68 to promote, in Ad-infected cells, the

exci-sion and replication of an ITR-flanked cassette contained in a

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FIG. 1. Structure, expression, and intracellular distribution of fusion constructs derived from wt Rep78 and Rep68. (A) Diagram of the different chimeras made up of full-length Rep78 or Rep68 fused with the PR891-HBD. For all constructs, the HBD was the same (residues 642 to 891 of the human progesterone receptor). For PR891-HBD, Rep78, and Rep68, the numbers above the diagrams refer to the amino acid positions. For the fusions, the numbers do not refer to the amino acid position in the context of the fusion protein but instead indicate the amino acid positions of the corresponding parental Rep protein. (B) Expression levels of the various fusions in transiently transfected HeLa cells. Whole-cell extracts were prepared from HeLa cells transfected with the expression vectors for wt Rep78, wt Rep68, and the six different chimeras. wt Rep78, wt Rep68, and their derivatives were detected by immunoblotting with a rabbit polyclonal serum against Rep proteins. Lane 9 contains untransfected cells. (C) Representative micrographs of the staining patterns observed in Hep3B cells transfected with Rep78, Rep68, and the various Rep/PR fusions. Hep3B cells were transfected with 10␮g of the expression vectors pCMV/Rep78, pCMV/Rep68, and pCMV/Rep68/PR. In this last case, cells were treated (w/ RU486) or not (w/o RU486) with 100 nM RU486. The cells were stained with a rabbit polyclonal antibody directed against the Rep moiety (see also Materials and Methods). The staining was classified into three categories: N, predominantly nuclear fluorescence; C, predominantly cytoplasmic staining; N⫽C, equal cytoplasmic and nuclear staining. At least 1,000 stained cells, obtained from a minimum of three experiments, were scored for each protein. The numbers below the micrographs represent the percentage of cells falling into each category.

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cotransfected plasmid (45). The plasmid ITR/Hook-Neo (30),

containing the expression cassette for the membrane-bound

single-chain antibody (Hook) and for the neomycin resistance

genes (

neo

) cloned between the AAV ITRs, was cotransfected

with expression vectors for the four Rep/PR fusions in

Ad-2-infected HeLa cells, treated or not treated with RU486.

Low-molecular-weight DNA was isolated 63 h posttransfection (20),

digested with

Dpn

I to degrade input plasmid DNA (62), and

fractionated by electrophoresis on an agarose gel. Monomeric

and dimeric forms of the rescued and replicated ITR-flanked

Hook-

neo

cassette were detected on Southern blots by using a

32

P-labelled

neo

probe: the intensity of the signal was

consid-ered to be a measurement of the activity of the various Rep/PR

fusions in the assay. The autoradiogram of one such

experi-ment is presented in Fig. 2B: Fig. 2C shows the corresponding

agarose gel, which was blotted onto a nylon membrane. No

sig-nal was detected in untransfected cells or in cells transfected

only with ITR/Hook-

neo

(Fig. 2B, lanes 1 and 2). In cells

trans-fected with expression vectors for the Rep78/PR, Rep78int/

PR, Rep68/PR, and Rep68int/PR chimeras, bands

correspond-ing to rescued monomers and dimers were clearly detected in

the absence of RU486 treatment (lanes 3, 5, 7, and 9); their

intensities were similar to those monitored in cells transfected

with wt Rep78 and Rep68 (lanes 11 and 12) and was not

in-creased following RU486 treatment (lanes 4, 6, 8, and 10).

Identical results were obtained with 293 and Hep3B cells (not

shown). In conclusion, we found that the four chimeric Rep/PR

proteins displayed a constitutive rather than

hormone-induc-ible activity in both the p5 promoter repression and

rescue-replication assays. This suggested that the relatively small

amount of protein present in the nuclei of untreated cells

(Table 1) was not only constitutively active but also

suffi-cient to give a full response in our experimental settings (see

also Discussion).

It is known that a constitutively nuclear fusion protein can be

rendered hormone responsive by placing the HBD in close

contact with the active domains of the heterologous protein

(40, 41). We thus decided to generate a new set of fusions in

which the distance of the HBD from the potentially regulatable

activities of Rep was reduced. To do this, a few Rep68 deletion

mutants were constructed to identify the minimal region of

Rep retaining wild-type activity and then fuse it with

PR891-HBD.

Identification of the minimal region of Rep68 retaining full

activity in vitro and in vivo.

Since the N terminus of Rep68 is

required for DNA binding, deletions were generated starting

from the carboxy terminus of the protein (37). Four

C-termi-nally truncated Rep68 derivatives were thus constructed,

name-ly, Rep1–520, Rep1–502, Rep1–491, and Rep1–484, which lack

the last 17, 35, 46, and 53 C-terminal amino acids, respectively.

Their schematic structure is shown in Fig. 3A. No further

deletions were constructed, because they have been previously

reported to strongly impair DNA binding and endonuclease

activity (34, 61, 67).

The four Rep68 derivatives were first analyzed in vitro.

Rep1–520, Rep1–502, Rep1–491, and Rep1–484, as well as

Rep68, were produced by in vitro translation and then assayed

for their DNA binding and endonuclease activity. As shown in

Fig. 3B and C, Rep1–520, Rep1–502, and Rep1–491 bound

(Fig. 3B) and cleaved (Fig. 3C) a 5

⬘

-end-labelled hairpinned

AAV ITR as efficiently as Rep68 did, while Rep1–484

dis-played a weaker activity in both assays (Fig. 3B and C).

Immunoblotting analysis demonstrated that all four mutants

were expressed at levels comparable to that of Rep68 in HeLa,

Hep3B, and 293 cells (results not shown). The major Rep

nu-clear localization signal (NLS) spans a region between amino

acids 485 to 510 which is highly enriched in positively charged

residues (Lys and Arg) (21, 51, 66): since three of four deletion

mutants lacked at least part of this sequence, we first checked

their intracellular distribution. As shown in Table 2,

progres-sive deletions from the C terminus of Rep68 gradually reduced

the capacity of the mutants to localize into the nucleus. Rep1–

520 behaved like Rep68 (N

⫽

95% [Table 2]), and Rep1–484,

which lacked the entire NLS, was found predominantly in the

cytoplasm of all scored cells (C

⫽

100% [Table 2]). Rep1–502

and Rep1–491 did not display a clear preferential subcellular

compartimentalization but were still capable of localizing into

the nuclei (Table 2).

The in vivo activity of the four mutants was then tested in

both the p5 promoter-repression and the rescue-replication

assays performed with various cell lines. As summarized in

Table 3, Rep1–520, Rep1–502, and Rep1–491 displayed a

wt-like activity in both tests. In contrast, Rep1–484 acted as a poor

repressor and was totally inactive in the rescue-replication

assay, in agreement with its reduced activity in vitro (Fig. 3B

and C) and its mainly cytoplasmic distribution (Table 2).

Fi-nally, we checked the integration competence of the various

Rep68 derivatives by using a recently developed PCR-based

integration assay (30). HeLa cells were cotransfected with ITR/

Hook-Neo and the various mutants and were then serially

passaged for 14 days in the absence of selection. ITR/AAVS1

junctions were then selectively amplified by PCR and revealed

by Southern blotting as described in the footnotes to Table 3

(30, 52, 55). As previously reported, under these experimental

conditions no signal is detected in cells transfected only with

the ITR/Hook-Neo cassette (reference 30 and data not shown). A

positive signal, indicative of site-specific integration events at

the AAVS1 site, was instead detected in cells in which plasmid

ITR/Hook-Neo was cotransfected with the expression vectors

for Rep78 or Rep68 and its derivatives Rep1–520, Rep1–502,

and Rep1–491 (Table 3). Conversely, no site-specific

integra-tion was observed when Rep1–484 was used (Table 3).

In summary, the experiments performed with Rep68

C-ter-minally deleted mutants demonstrated that Rep1–491 is the

shortest variant which retains the capacity to localize into the

nuclei, although it does so less efficiently than wt protein, while

still displaying full in vitro and in vivo activity, including

site-specific integration. However, Rep1–484 binds and nicks DNA

with reduced efficiency in vitro and is poorly active in vivo, also

because it lacks a functional NLS.

TABLE 1. Effects of RU486 on the intracellular distribution of

full-length Rep78 and Rep68 fused with PR891-HBD

Protein _treatmentRU486a

Intracellular localization (%)b

N N⫽C C

Rep78 and Rep68

⫺

95

5

0

Rep78/PR

⫺

⫹

90

1

7

6

92

4

Rep78int/PR

⫺

⫹

92

2

4

3

94

5

Rep68/PR

⫺

⫹

91

2

6

4

92

5

Rep68int/PR

⫺

⫹

91

1

4

4

95

5

a_{⫺, transfected cells were untreated;⫹, 100 nM RU486 was added to the}

culture medium.

b_{Stained Hep3B cells were classified as described in the legend to Fig. 1C. At}

least 1,000 stained cells, obtained in three or more experiments, were analyzed for each protein, and the results are expressed as the percentage of cells included in each category.

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Construction of Rep1–484/Pn and Rep1–491/P.

In an

at-tempt to generate a hormone-dependent Rep protein, we

con-structed two new Rep/PR hybrid proteins (Fig. 4A). In the

first, Rep1–491/P, the PR891-HBD was fused to the C

termi-nus of Rep1–491. The second chimera, named Rep1–484/Pn,

was generated by C-terminally fusing Rep1–484 to a slightly

larger segment of the human PR (aa 635 to 891), which

in-cludes the major NLS (aa 638 to 642) of the human PR (16,

FIG. 2. Activity of chimeras derived from wt Rep78 and Rep68. (A) Rep/PR fusions constitutively inhibit p5 promoter activity. 293 cells were transfected with 5

␮g of plasmid p5/LUC and 50 ng of the expression plasmids pCMV/Rep78, pCMV/Rep68, pCMV/Rep78/PR, pCMV/Rep78int/PR, pCMV/Rep68/PR, and pCMV/ Rep68int/PR (see Materials and Methods). In control experiments, the empty expression vector pcDNAIII was cotransfected with p5/LUC. At 15 h posttransfection, the cells were treated for 36 h or not treated with RU486. The luciferase activity observed in the presence of the different Rep and Rep derivative expression vectors was calculated as the percentage of that (arbitrarily assumed to be 100%) measured in cells transfected with p5/LUC and pcDNAIII. White columns show activity in the absence of RU486 treatment; black columns show activity in the presence of 100 nM RU486. Each column represents the mean and standard deviation for at least three different experiments, performed in duplicate with different plasmid preparations. (B) Constitutive activity of Rep/PR fusions in a rescue-replication assay. Ad-2-infected HeLa cells were cotransfected with 10␮g of the ITR/Hook-Neo plasmid and 10␮g of the expression plasmids pCMV/Rep78 (lane 11), pCMV/Rep68 (lane 12), pCMV/Rep78/PR (lanes 3 and 4), pCMV/Rep78int/PR (lanes 5 and 6), pCMV/Rep68/PR (lanes 7 and 8), or pCMV/Rep68int/PR (lanes 9 and 10). After 15 h, the cells were washed and incubated either with normal medium (⫺) or with medium containing 100 nM RU486 (⫹). After 48 h, low-molecular-weight DNA samples were isolated (20), digested withDpnI (62), and analyzed on Southern blots with a32_{P-labelledneo-derived probe. The two bands corresponding to rescued}

monomeric (about 3.7-kb) and dimeric (about 7.5-kb) ITR-flanked cassette are visible. Higher-order multimeric forms were evident after longer exposures (data not shown). In control experiments, cells were transfected only with the ITR/Hook-Neo plasmid (lane 2). Untransfected cells are shown in lane 1. Lane 13 shows results obtained in cells cotransfected with 10␮g of plasmid ITR/Hook-Neo and 10␮g of plasmid pCMV/Rep, which express all four species of Rep (see also Materials and Methods). Molecular sizes are shown in kilobases. The autoradiogram shown is representative of five different experiments which all gave similar results. (C) Ethidium bromide staining of the agarose gel which was blotted onto a nylon membrane. Numbering below the lanes is the same as in B.

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69); this motif was expected to facilitate the intranuclear

lo-calization of the fusion.

Rep1–484/Pn and Rep1–491/P were cloned into eukaryotic

expression vectors; their expression levels in transfected HeLa,

293, and Hep3B cells were comparable to that of wt Rep68

(results not shown). Immunofluorescence experiments

re-vealed that in untreated cells, both fusions were on average

predominantly cytoplasmic or evenly distributed between the

nucleus and cytoplasm (Fig. 4B). RU486 modestly increased

the intranuclear accumulation of Rep1–484/Pn, whereas a

more pronounced effect was observed for Rep1–491 (Fig. 4B).

In this last case, upon hormone treatment there was a

substan-tial increase in the number of cells stained exclusively in the

nuclei (N

⫽

20% [Fig. 4B]) and a drastic reduction in the

number of cells in which Rep1–491/P was localized only in the

cytoplasm (C

⫽

71 and 10% in the absence and presence of

FIG. 3. Structure and in vitro activity of Rep C-terminal deletion mutants. (A) Schematic representation of the Rep deletion mutants. Numbers to the right refer to amino acid positions. (B) Electrophoretic mobility shift assay with wt and mutant Rep proteins. Rep68 and the four deletion mutants were translated in vitro, and equivalent amounts of the various proteins (normalized as described in Materials and Methods) were used in dose-dependent DNA binding assays. Reaction mixtures contained 20,000 cpm of32_{P-5⬘-end-labeled AAV ITR and either no protein (lane 1) or increasing concentrations of the various proteins indicated above lanes 2 to}

21. (C) Nicking activity of wild-type and mutant Rep proteins. A 20,000-cpm sample of32_{P-5⬘-end-labeled AAV ITR containing a single-stranded trs (trs}⫹_{[48]) was}

incubated with increasing concentrations, normalized as in panel B, of in vitro-translated Rep68, Rep1–484, Rep1–491, Rep1–502, and Rep1–520. A standard endonuclease reaction was carried out (30, 48), and the reaction products were resolved on an 8% polyacrylamide sequencing gel. The positions of the substrate (trs

⫹) and of the released 73-bp fragment are indicated. The two labelled fragments shorter than 73 bp are the result of aberrant nicking sometimes observed when single-strandedtrs⫹_{substrates are used in Rep endonuclease assays (30, 48).}

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RU486, respectively [Fig. 4B]). Similar results were obtained

with Hep3B (Fig. 4B), HeLa, and 293 (results not shown) cells.

Ligand-dependent activity of Rep1–484/Pn and Rep1–491/P.

The two fusions were first tested for their capability to

down-regulate the p5 promoter activity in 293 cells. As shown in Fig.

5A, both fusions displayed low repressing activity in the

ab-sence of the hormone (about 20% repression [Fig. 5A]):

how-ever, following RU486 treatment, Rep1–491/P strongly

inhib-ited p5/LUC activity (94% repression [Fig. 5A]). The

repressing activity of Rep1–484/Pn was also stimulated by the

steroid but to a more limited extent (45% repression [Fig.

5A]). Similar results were obtained with HeLa cells (not

shown).

Rep1–484/Pn also displayed hormone dependence in the

rescue-replication assay: in fact, rescue-replication of the

ITR-flanked Hook-

neo

cassette was seen in Ad2-infected HeLa

cells only upon RU486 treatment (Fig. 5B, compare lanes 2

and 3). However, the maximal activity was lower than that of wt

Rep68 (compare lanes 3 and 6). Also, Rep1–491/P was at least

partially hormone dependent in this assay: in fact, basal activity

was detectable in the absence of RU486 (lane 4), but the

activity was strongly enhanced by steroid treatment (compare

lanes 4 and lane 5). The same results were observed with 293

and Hep3B cells (not shown).

The capability of Rep1–484/Pn and Rep1–491/P to mediate

site-specific integration was then examined by using the

PCR-based assay described above. As shown in Fig. 5C, no

site-specific integration could be monitored in cells transfected

with Rep1–484/Pn (Fig. 5C, lanes 6 and 7). In contrast, Rep1–

491/P clearly triggered integration at AAVS1 in the presence

of RU486 (lanes 4 and 5). It is worth noting that in this assay,

the full-length Rep proteins fused with PR891-HBD also

dis-played a constitutive activity (data not shown). The

heteroge-neous size of the amplified AAVS1-positive bands is in

agree-ment with the fact that Rep-mediated integration occurs in a

region spanning more than 500 bp of human chromosome 19

(30, 44, 46, 65). To further verify that positive signals are

indicative of true integration events, we cloned two junctions

amplified from hormone-treated cells transfected with Rep1–

491/P. Their sequences, shown in Fig. 5D, demonstrate that

integration has occurred at nucleotides 842 and at 950 of

AAVS1. In both cases, a complete ITR was not found, in line

with all the ITR/AAVS1 junctions analyzed so far (30, 38, 42,

43, 46, 65).

Rep1–491/P mediates site-specific integration in the absence

of chromosomal rearrangements.

To better characterize the

integration efficacy of Rep1–491/P, we performed Southern

blot analysis of individual clones derived from HeLa cells

co-transfected with the expression vector for Rep1–491/P and

plasmid ITR/Hook-Neo. At 15 h posttransfection, the cells

were incubated for 24 h with 100 nM RU486 and then grown

in the absence of steroid treatment under selection with 700

␮

g

of G418 per ml for 3 weeks. Genomic DNA was then extracted

from individual clones and digested with the restriction

en-zyme

Bam

HI, which has no recognition site in the ITR-flanked

Hook-

neo

cassette and in the region of AAVS1 in which the

great majority of integration events take place (27, 30, 46, 65).

Digested DNA was then subjected to Southern blot analysis

with AAVS1-derived and

neo

-derived probes. Site-specific

in-tegration was assigned to clones displaying an AAVS1

hybrid-izing band which was upshifted with respect to the bands

ob-served in untransfected cells and which also cohybridized with

the

neo

probe (30).

According to this criterion, site-specific integration was

ob-served in 7 of 28 clones (25% frequency of site-specific

inte-gration) derived from cells cotransfected with ITR/Hook-Neo

and the expression vector for Rep68. This integration efficiency

is in line with previously published results (30). Also, Rep1–

491/P was able to mediate integration at the AAVS1 site, and

the frequency of site-specific integration was higher in the

presence of RU486 treatment (25%; 7 positive clones of 28

analyzed) than in its absence (3.2%; 1 positive clone of 31),

confirming that the activity of the fusion was in large part

under hormonal control. Of the clones derived from

Rep68-transfected cells, 40% showed additional AAVS1 bands not

cohybridizing with the

neo

probe. As illustrated in Fig. 6, where

the integration pattern of some representative clones is shown,

these

neo

-negative bands were evident both in clones scored as

positive for site-specific integration (Fig. 6, lanes 2 and 6) and

in those scored as negative (lanes 3 and 5). Interestingly,

AAVS1-positive and

neo

-negative bands were not observed in

any of the clones derived from cells transfected with Rep1–

491/P and treated or not treated with RU486 (lanes 7 to 12 and

data not shown). These results suggest that short-term (24-h)

treatment with RU486 enables Rep1–491/P to promote

site-specific integration as efficiently as wt Rep68 but with much

TABLE 2. Intracellular distribution of Rep C-terminal

deletion mutants

Protein Intracellular localization (%)

a

N N⫽C C

Rep1–520

95

5

0

Rep1–502

57

42

1

Rep1–491

16

42

42

Rep1–484

100

a_{Stained Hep3B cells were classified as described in the legend to Fig. 1C. A}

total of 600 stained cells, obtained from at least two different experiments, were analyzed for each protein, and the results are expressed as the percentage of cells included in each category.

TABLE 3. Activity of Rep68 C-terminal deletion mutants

in transfected cells

Protein _{p5/LUC activity (%)}Repression of a _replicationRescue-b Integrationc

Rep78

98

⫾

1

⫹

⫹

Rep68

98

⫾

1

⫹

⫹

Rep1–520

97

⫾

2

⫹

⫹

Rep1–502

98

⫾

1

⫹

⫹

Rep1–491

95

⫾

4

⫹

⫹

Rep1–484

40

⫾

3

⫺

⫺

a_{Repression of p5/LUC activity was analyzed as described in the legend to Fig.}

2A. 293 or HeLa cells were cotransfected with plasmid p5/LUC and the expres-sion vectors for the various Rep derivatives. Represexpres-sion was calculated with respect to the luciferase activity observed in extracts from cells transfected with p5/LUC plasmid alone.

b_{Rescue-replication of the Hook-neocassette in Ad2-infected HeLa, 293, or}

Hep3B cells was monitored as described in the legend to Fig. 2B.⫹, detection of a rescue-replicated Hook-Neo cassette in the Hirt supernatant of transfected cells (the intensity of the signal was identical for all the active mutants);⫺, no replicated cassette could be detected. Similar results were obtained in all three cell lines.

c_{HeLa cells were cotransfected with the expression vectors for the various}

mutants and plasmid ITR/Hook-Neo and then serially passaged for 14 days in the absence of selection. Genomic DNA was extracted and used as a template for two consecutive rounds of PCR with nested primers specific for the AAV ITRs and the AAVS1 site in human chromosome 19 to selectively amplify the ITR/ AAVS1 junctions (30). Amplification products were then detected by Southern blot analysis with an AAVS1-derived probe (30, 52, 55).⫹and⫺indicate that AAVS1-positive signals were observed or not, respectively.

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less propensity to generate additional and undesired

rear-rangements at the AAVS1 site.

We finally checked whether the stable transformants scored

as positive for site-specific integration also expressed the other

gene, Hook, contained between the AAV ITRs in plasmid

ITR/Hook-Neo. As shown in Fig. 7, Western blot analysis

revealed that all seven stable transformants derived from cells

transfected with Rep1–491/P also expressed the Hook gene

FIG. 4. Structure and intracellular distribution of chimeras made up of Rep68 C-terminal deletion mutants fused with PR891-HBD. (A) Diagram of Rep1–491/P and Rep1–484/Pn chimeric constructs. The region of the human PR spanning from aa 635 to 891 is shown at the top of the figure: the NLS (aa 635 to 642), which is maintained in the Rep1–484/Pn fusion but absent in the Rep1–491/P chimera, is indicated in black. Numbers above the hybrid proteins refer to the amino acid position in the parental Rep protein (see also the legend to Fig. 1A). (B) RU486 affects the intracellular distribution of Rep1–491/P and Rep1–484/Pn. Hep3B cells were transfected with 10␮g of the expression vectors pCMV/Rep1–491/P and pCMV/Rep1–484/Pn and treated with 100 nM RU486 or left untreated. The cells were stained with an anti-Rep polyclonal serum and classified as described in the legend to Fig. 1C. At least 1,000 stained cells, obtained from a minimum of three experiments, were scored for each fusion. The numbers below the micrographs represent the percentage of cells falling into each category.

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protein product (sFv/PDGFR) (6). Conversely, the Hook gene

protein product was detectable in only one of the seven

AAVS1 integrants isolated from cells cotransfected with wt

Rep68 (results not shown). This finding further suggests that

Rep1–491/P promotes a more precise integration of an

ITR-flanked cassette at the AAVS1 site.

DISCUSSION

In this paper we describe the construction and

characteriza-tion of a ligand-dependent AAV Rep protein. Among the two

ligand-dependent Rep/PR fusions generated, Rep1–491/P has

the most interesting properties in that in various assays it

displays a low basal activity which is highly stimulated by

RU486 and, more importantly, promotes integration into the

AAVS1 site in a ligand-dependent manner. The partial

dis-crepancy in the results that emerged from the PCR-based

integration assay (no junctions amplified from pooled and

un-selected cells in the absence of RU486 treatment [Fig. 5C, lane

4]) and the Southern blot analysis of individual selected clones

(site-specific integration detected in 1 of 31 clones derived

from untreated cells) might simply reflect different sensitivities

of the two assays. Nevertheless, further work is required to

clarify whether selection can increase the integration

fre-quency even in the absence of hormone treatment.

Rep1–491/P has several advantages over wt Rep78 or Rep68

for gene therapy purposes. This chimera triggers site-specific

integration in the presence of RU486 as efficiently as wt Rep68

does, and it does so without generating major unwanted

rear-rangements at the AAVS1 site, thus overcoming one of the

major limitations of the AAV-based integration strategy (31).

FIG. 5. Hormone-dependent activity of Rep1–491/P and Rep1–484. (A) Rep1–491/P and Rep1–484/Pn repress the p5 promoter in a ligand-dependent manner. 293 cells were transfected with 5␮g of plasmid p5/LUC and 50 ng of the expression plasmids pCMV/Rep68, pCMV/Rep1–491/P, and pCMV/Rep1–484/Pn. Luciferase activity was calculated as described in the legend to Fig. 2A. White and black columns represent the activities measured in the absence and in the presence of 100 nM RU486, respectively. Each column represents the mean and standard deviation for at least three different experiments, performed in duplicate with different plasmid preparations. (B) RU486 stimulates the activity of Rep1–491/P and Rep1–484/Pn in a rescue-replication assay. Ad2-infected HeLa cells were cotransfected with 10␮g of the ITR/Hook-Neo plasmid and 10␮g of the expression plasmid pCMV/Rep68 (lane 6), pCMV/Rep1–491/P (lanes 4 and 5), or pCMV/Rep1–484/Pn (lanes 2 and 3). Cell treatment and analysis of low-molecular-weight DNA was performed as described in the legend to Fig. 2B. Monomeric (about 3.7-kb) and dimeric (about 7.5-kb) forms of the rescued ITR-flanked cassette are visible: higher-order multimeric forms were detectable after longer exposures (data not shown). In control experiments, the ITR/Hook-Neo plasmid was cotransfected with the empty expression vector pcDNAIII (lane 1). Molecular sizes are shown in kilobases. The autoradiogram shown is representative of four different experiments which all gave similar results. (C) RU486-dependent site-specific integration mediated by Rep1–491/P. HeLa cells were transfected with 10␮g of plasmid ITR/Hook-Neo alone (lane 2) or together with 10␮g of the expression vector pCMV/Rep68 (lane 3), pCMV/Rep1–491/P (lanes 4 and 5), or pCMV/Rep1–484/Pn (lanes 6 and 7). At 15 h later, the cells were washed and incubated for 24 h with 100 nM RU486 or left untreated. ITR/AAVS1 junctions were amplified from the genomic DNA extracted from cells subcultured for 14 days and detected with an AAVS1-derived probe as described in the footnote to Table 3. Lane 1 contains untransfected cells. Molecular sizes are shown in base pairs. (D) Sequence analysis of ITR/AAVS1 junctions. The letters D and A refer to the accepted nomenclature for AAV/ITR sequences (4, 30, 46). The numbers above the diagrams refer to the last identifiable viral and AAVS1 nucleotides. Insertions between AAV/ITR and AAVS1 are boxed. AAVS1 breakpoints are based on published AAVS1 sequence (27). Nucleotide numbering of the AAV/ITR is relative to the right end of the AAV genome (51).

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It is not yet clear how rearrangements are generated and

whether they occur at the same time as or after the initial

integration event (33), but our results with Rep1–491/P

indi-cate that it is possible to prevent or at least reduce them by

simply establishing a time limit to Rep activity in target cells.

These results tempt us to speculate that rearrangements at

AAVS1 are not a direct consequence of the integration process

itself but, rather, might be ascribed to unrestrained activity of

Rep at the integration locus. This is further corroborated by

the recent report that AAVS1 rearrangements are also

de-tected in cells transfected with a plasmid containing the entire

rep

ORF only and no ITR-flanked sequences (52; S. Lamartina

and C. Toniatti, unpublished results).

We might hence envision the following scenario: after the

initial integration event, rearrangements occur when

constitu-tively active Rep78/68 binds and nicks its target sequence

present not only at AAVS1 but also in the ITRs flanking the

integrated transgene. This leads to partial replication,

rear-rangements, and, possibly, translocation of the region (2, 31,

32, 52). In relation to this last point, it is of interest that the

Rep78 or Rep68 nicking sites located on the ITRs are

main-tained in all the AAVS1-ITR junctions sequenced so far and

that in the majority of cases, the ITRs flanking the integrated

DNA still retain their Rep binding site (30, 38, 42, 43, 46, 65).

In contrast, Rep1–491/P is capable of triggering site-specific

integration during the initial 24 h of RU486 treatment of

transfected cells; following withdrawal of the hormone, it no

longer binds and nicks DNA, thus reducing the instability of

the region.

The two genes, Hook and

neo

, contained between the AAV

FIG. 6. Southern blot analysis of HeLaneo-resistant clones derived from cells cotransfected with plasmid ITR/Hook-Neo and expression vectors for either wt Rep68 or Rep1–491/P. Transfection and selection of Neor_{clones were carried out as described in Materials and Methods. Genomic DNA of isolated clones was digested with} BamHI and blotted onto a nylon membrane. (A) Hybridization to an AAVS1-specific probe. (B) The same membrane after rehybridization to aneo-specific probe. Solid triangles mark upshifted bands which cohybridize with both probes and are therefore indicative of site-specific integration (panels A and B, lanes 2, 6, 8, 9, 11, and 12). Open triangles show nonspecific rearrangements (AAVS1-positive/neo-negative bands) observed in clones derived from cells cotransfected with wt Rep68 (panel A, lanes 2, 3, 5, and 6). cR68, clones derived from cells transfected with wt Rep68 (lanes 1 to 6); cRP⫹, clones derived from cells transfected with Rep1–491/P and treated for 12 h with 100 nM RU486 (lanes 7 to 12). Lane 13 contains untransfected cells. Molecular sizes are shown in kilobases.

FIG. 7. Hook gene expression in site-specific integrants derived from HeLa cells cotransfected with ITR/Hook-Neo and Rep1–491/P and treated with RU486. Whole-cell extracts were prepared from cRP⫹clones and run on an SDS-polyacrylamide gel. Fractionated proteins were transferred to a nitrocellu-lose membrane, which was probed with anti-mycepitope tag antibody (13) as described in Materials and Methods. The arrow marks the band, of the expected size, corresponding to the single-chain antibody (sFv/PDGFR) encoded by the Hook gene (6). Asterisks indicate nonspecific product recognized by the anti-myc monoclonal antibody 9E10.2 in untransfected cells. Lane 8 contains untrans-fected HeLa cells.

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ITRs were both expressed in all site-specific integrants derived

from hormone-treated cells transfected with Rep1–491/P (Fig.

7). In addition, in six of seven clones the size of the single

upshifted AAVS1 band are about 6.5 kb long (Fig. 6A, lanes 8,

9, and 12, and data not shown), a size which is consistent with

the integration of one single copy of the full-length

ITR/Hook-Neo cassette (3.7 kb). These findings are in striking contrast to

what was observed, in this and previous studies, in site-specific

stable integrants derived from cells cotransfected with wt

Rep68 (Fig. 6A) (2, 47, 52). We should mention that

integra-tion of the Hook gene at AAVS1 in our selected clones could

not be unequivocally demonstrated by Southern blotting,

be-cause the Hook-derived probe reveals a complex pattern of

multiple bands on genomic DNA (Hook codes for a

single-chain antibody [6]). The use of different ITR-flanked cassettes

will therefore be required for a careful study of the fine

struc-ture of the integrated DNA. This will be the object of fustruc-ture

work to clearly establish whether Rep1–491/P not only reduces

nonspecific rearrangements but also can promote a more

pre-cise integration of the ITR-flanked cassette.

Shorter variants of Rep (Rep1–491 and Rep1–484) fused

with PR891-HBD proved hormone dependent, while fusions

with full-length Rep78 or Rep68 did not. The observation that

tighter hormonal control can be achieved by reducing the

dis-tance between HBD and the active site of the heterologous

moiety is not unprecedented (35, 40, 50). Nevertheless, the

precise mechanisms by which heterologous proteins fused with

HBDs are regulated by the cognate ligand have not yet been

elucidated and are likely to vary according to the particular

steroid receptor used (41, 54). A point to note is that in our

case, the more stringent control achieved with the shorter

fusions did not parallel a concomitant tighter regulation of

their subcellular distribution. In fact, both fusions with Rep

deletions and fusions with full-length Rep78/68 were

predom-inantly cytoplasmic, presumably complexed with heat shock

protein 90 (Hsp90) (49, 54), in the absence of RU486 and were

localized into nuclei following hormone treatment. It is

con-ceivable that the constitutive activity of full-length Rep

pro-teins fused with PR891-HBD results from the highly sensitive

assays we used to monitor Rep activity in transfected cells.

Furthermore, immunofluorescence experiments gave a static

representation of what is probably a dynamic situation in

which, similarly to sex steroids such as progesterone, estrogen,

and glucocorticoid receptors (54), a specific Rep/PR fusion

continuously shuttles between the nucleus and the cytoplasm

with, at any given time, a major fraction of the protein being

localized in one of the two compartments (28, 54).

Neverthe-less, our results suggest that regulation of intranuclear

local-ization is probably neither the only nor the most important

mechanism responsible for the ligand-dependent activity of

Rep1–491/P and Rep1–484/Pn. Although we have no data to

support this hypothesis, it is tempting to speculate that the

major difference between full-length Rep/PR and deleted

Rep/PR fusions is that only the latter require RU486 to

as-sume the proper conformation, thus acquiring full activity

and/or the ability to interact with specific intracellular proteins.

In relation to this point, it has to be remembered that the

progesterone receptor is predominantly nuclear, regardless of

the presence of its ligand, but interacts with appropriate

co-factors and stimulates transcription only in the presence of the

hormone (28, 36, 64).

Rep78 and Rep68 repress the AAV p5 promoter: this

re-pression is postulated to be mediated in part by direct binding

to the p5 RBS and in part by interaction with as yet

unidenti-fied cellular factors that might facilitate repression (29, 39).

Rep1–491/P efficiently down-regulated the p5 promoter in a

hormone-dependent manner, although background activity

(20% repression [Fig. 5A]) was also observed in the absence of

RU486 treatment. It is worth noting that p5 promoter

repres-sion is an extremely sensitive test, as demonstrated by the

behavior of mutant Rep1–484, which is unable to promote

rescue-replication and to drive integration at AAVS1 but is

still active in this assay (21). Further investigation is required

to assess the activity of Rep1–491/P on heterologous

promot-ers, but it is reasonable to expect that the chimeric protein,

which is localized mainly in the cytoplasm in the absence of

hormone treatment, should prove less capable than Rep78/68

of interfering with the expression of cellular genes and,

ulti-mately, with cellular physiology. This hypothesis is further

sup-ported by the evidence that when transfected in 293 cells,

Rep78 reduced their cloning efficiency by 80% while Rep1–

491/P had only a modest effect in the absence of RU486

treat-ment (D. Rinaudo and C. Toniatti, unpublished results). These

results complement previous reports indicating that the growth

rate of 293 cells stably expressing an inducible Rep is altered

and reduced in the presence of the inducer (68).

An interesting property of Rep1–491/P is that it is not

re-sponsive to endogenous steroids such as progesterone, thus

rendering the use of this protein feasible not only for ex vivo

but also for in vivo gene therapy. It has been recently reported

that Rep-mediated site-specific integration at AAVS1 takes

place in transgenic rodents (mice and rats) carrying the human

AAVS1 site (43), and it would be of interest to test the

inte-gration competence of Rep1–491/P in this animal model. The

ideal vector for in vivo utilization of Rep1–491/P and, in

gen-eral, of the AAV integration machinery, has yet to be

con-structed, but one possible approach is that of introducing both

an ITR-flanked transgene and a Rep1–491/P expression

cas-sette into an Ad vector. This Ad/AAV chimeric virus would

borrow properties from both Ad vectors (i.e., infectivity, high

titer, and large capacity) and AAV (integration competence).

We have recently constructed a helper-dependent Ad vector

containing the Rep78 gene and demonstrated that this

chi-meric Ad/AAV vector is indeed capable of triggering

site-specific integration of a codelivered ITR-flanked cassette in

cultured cells (42). A further step in this direction will be to use

the ligand-dependent Rep1–491/P for the generation of

addi-tional Ad/AAV hybrid vectors to be tested in AAVS1

trans-genic rodents.

ACKNOWLEDGMENTS

We are grateful to B. O’Malley and S. Tsai for PR891-HBd cDNA.

We also thank Janet Clench for editing the manuscript and M. Emili

for contributing graphical work.

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