The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo

Avolio F

Chadeuf G

François A

Guilbaud M

Moullier P

RECCHIA, Alessandra

Salvetti A.

English

François A

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

JOURNAL OF VIROLOGY, Mar. 2008, p. 2590–2593 Vol. 82, No. 50022-538X/08/$08.000 doi:10.1128/JVI.01956-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5Element for Recombinant AAV Vector Site-Specific IntegrationMickae¨l Guilbaud,1,2 Gilliane Chadeuf,1 Fabio Avolio,3 Achille Franc¸ois,1 Philippe Moullier,1,2Alessandra Recchia,3,4 and Anna Salvetti1,5*INSERM, U649, Nantes F-44000, France1; EFS des Pays de la Loire, Nantes F-4400, France2; Istituto Scientifico H. San Raffaele,Gene Therapy Program, Milano, Italy3; University of Modena and Reggio Emilia, Department of Biomedical Sciences,Modena, Italy4; and INSERM, U758, Lyon F-69007, France5Received 6 September 2007/Accepted 12 December 2007The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essentialfor Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We reporthere that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does notsignificantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested orproliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectorsin vivo.Adeno-associated virus type 2 (AAV-2) was originally foundto be able to integrate into the AAVS1 locus of human chro-mosome 19 in a site-specific manner (6a), and since then,several studies have been conducted that confer this capacity torecombinant AAV (rAAV) vectors. It was recently reportedthat a 138-bp p5 region (nucleotides 151 to 289) of the AAV-2genome is an essential cis-acting element required for Rep-mediated site-specific integration (RMSSI) of plasmids, bothin the presence and in the absence of the viral inverted termi-nal repeats (ITRs) (10, 11). This viral region, which includesthe promoter of the rep68/78 gene, was previously character-ized as a cis-acting replication element (8, 9, 13), and recentanalyses defined a 55-bp p5 region (p5D10) as a minimal rep-lication element. This region contains the TATA box, the Repbinding site, the terminal resolution site, the Yin Yang 1(YY1) binding site located at position 1, YY1 and a down-stream 17-bp sequence that could potentially form a hairpinstructure (HP) with to the terminal resolution site at the top ofthe loop (3). These studies also indicated that the TATA boxflanking the Rep binding site was absolutely required for invivo replication of the p5 element, likely through TATA bind-ing protein-mediated enhancement of Rep-dependent bindingand nicking. Similarly, Murphy et al. recently confirmed thatmutating the TATA box severely compromised replication ofthe p5 region and also found that RMSSI of p5-containingplasmids was sensitive to the same mutations as those affectingreplication of this element (7). However, these studies, like theprevious ones, were performed by analyzing stable cell clonesderived from actively dividing cells, such as HeLa or 293 cells,transfected with p5-containing plasmids. Therefore, the funda-mental question that remained to be answered concerned theeffect of the p5 element on RMSSI in the context of the ITRsand during viral infection of human cells.Validation of the minimal 55-bp p5 element using a rAAVplasmid-based assay. A first study was conducted to determineif the minimal 55-bp p5 element, identified as an efficientreplication origin (3), was also able to increase RMSSI ofrAAV plasmid as previously documented for the entire p5region. To answer this question, we used the procedure thatwas previously employed by other investigators (7, 10, 11),consisting of the analysis of stable cell clones isolated fromHeLa cells transfected with the rAAV plasmid containing dif-ferent versions of the p5 element inserted between the ITRs(Fig. 1). The wild-type (wt) 163-bp p5 region covers the entirep5 promoter from the major late transcription factor bindingregion to position 353, downstream from the HP region. The* Corresponding author. Mailing address: INSERM U758, EcoleNormale Supe´rieure de Lyon, 46 Alle´e d’Italie, F-69007 Lyon,France. Phone: 33 472728401. Fax: 33 472728137. E-mail: anna.salvetti@ens-lyon.fr. Published ahead of print on 19 December 2007.FIG. 1. Schematic view of the pAAV-GFP plasmids containing dif-ferent versions of p5. The star indicates the presence of a mutatedTATA box, and HP indicates the position of the putative HP (3).Positions refer to the wt AAV-2 genome (GenBank no. NC_001401).The control pAAV-GFP plasmid contains a nonrelevant 150-bp regioninstead of the p5 element.2590minimal 55-bp p5 region (p5D10) includes only the four ele-ments previously described as essential for in vivo and in vitroreplication (Fig. 1) (3). Since our previous analysis indicated acritical role of the TATA box for in vivo replication, two addi-tional replication-defective p5 elements containing either a mu-tation (p5mTATA) or a deletion (p5D12) of this binding sitewere inserted into the rAAV vector plasmid. Each rAAV plas-mid was cotransfected into HeLa cells together with a plasmidexpressing Rep under the control of doxycycline-inducible pro-moter (pBGTetOCMVRepTS) (12). Two weeks after trans-fection and culture in the presence of doxycycline, green flu-orescent protein (GFP)-positive cells were sorted by afluorescence-activated cell sorter and cloned by limiting dilu-tion. After expansion, each clone was analyzed for the pres-ence of the gfp sequence by PCR and visualization of GFPexpression. As expected, only a portion of the selected cloneshad integrated the gfp sequence (Table 1), and all the clonescontaining this gene also expressed the protein, although atdifferent levels (data not shown). In each of these GFP-stablecell clones, RMSSI was detected by Southern blot analysisperformed for EcoRI-digested DNA by using, first, an AAVS1probe to detect rearrangements of this chromosomal regionand then, after dehybridization of the membrane, a gfp probeto detect vector-containing bands (Fig. 2). Site-specific inte-gration of the rAAV vector plasmid was defined by the colo-calization of the gfp and AAVS-1 bands, as in previous studiesof RMSSI. As expected from previous studies, the presence ofthe wt p5 element significantly increased site-specific integra-tion of the rAAV plasmid compared to results with the pAAV-GFP vector (Table 1). Insertion of the minimal p5D10 elementalso had a positive effect on RMSSI, similar to that observedfor the entire p5 region. In this context, the use of long or shortreplication-defective p5 elements (p5mTATA and p5D12, re-spectively) also resulted in an increase of RMSSI, although ata level that was apparently lower than that observed with theirrespective wt counterparts. Altogether, the results of this plas-mid-based assay indicate that the minimal 55-bp region canincrease RMSSI of the rAAV plasmid to a level similar to thatobserved with the wt p5 region and that replication of thiselement is not strictly required to exert this activity.Effect of the wt and minimal p5 element during viral infec-tion of human cells. We next asked whether an effect of the p5element on RMSSI could similarly be observed during viralinfection of human cells. To determine this, pAAV-GFP plas-mids containing or not containing the p5 element were used toproduce rAAV particles using a rep-expressing plasmid withthe cytomegalovirus promoter substituting for the p5 promoterto avoid any recombination events with the p5-containingrAAV genome. The first model used for this study was com-posed of human hepatocyte-like cells derived after differenti-ation of the HepaRG hepatoma cell line (1, 4). This model waschosen because it represented human growth-arrested cellsdisplaying a quasi-normal karyotype, in particular concerningchromosome 19, thus resembling conditions found during wtand rAAV infection in vivo. For the experiments describedbelow, the cells were infected with rAAV-GFP particles 15days after being cultured in conditions inducing differentiation(4). The expression of Rep was achieved by coinfection with agutless adenovirus expressing the rep78 gene under the controlof a doxycycline-inducible promoter (Ad-Rep) (12). Quantita-tive PCR was then used to analyze RMSSI of each rAAVvector in differentiated HepaRG cells, 5 days postinfection.FIG. 2. Representative Southern blot analysis of RMSSI in stably transfected HeLa cells clones. HeLa cells (He) were cotransfected with eachpAAV-GFP plasmid (Fig. 1) and with a second construct encoding Rep78 under the control of a doxycycline-inducible promoter (pBGTetOCMVRepTS) (12). Whole genomic DNA extracted from of each cell clone was digested by EcoRI and analyzed by Southern blotting using anAAVS1 probe and, after dehybridization, a gfp probe. Genomic DNA from HA-16 cells and the pAAV-GFP plasmid (GFP) served as positivecontrols for AAVS1 disruption and gfp probe hybridization, respectively. The black arrow indicates the expected EcoRI restriction fragmentcontaining the AAVS1 sequence. The white arrows indicate AAVS1-disrupted bands that colocalize with gfp-containing bands.TABLE 1. Analysis of HeLa cell clones transfected with AAV-GFP plasmidsVector plasmid Total no.of clonesNo. ofGFP-positiveclonesa (%)No. of clones with AAVS1rearrangedb/no. of GFP-positive clones (%)No. of clones withRMSSIc/no. ofGFP-positiveclones (%)pAAV-GFP 87 17 (19) 5/17 (29) 1/17 (6)pAAVp5wt-GFP 81 32 (39) 15/32 (47) 12/32 (37)pAAVp5mTATA-GFP 64 15 (23) 8/15 (53) 3/15 (20)pAAVp5D10-GFP 71 19 (27) 8/19 (62) 5/19 (26)pAAVp5D12-GFP 85 40 (48) 14/40 (35) 5/40 (12)a As determined by PCR analysis and GFP expression.b As determined by Southern blotting using an AAVS1 probe.c Number of clones with AAV sequences integrated in AAVS1 region as determined by Southern blot analysis using gfp and AAVS1 probes.VOL. 82, 2008 NOTES 2591This assay, previously described by Hu¨ser et al. (5, 6), allowedthe detection of rAAV vector integration in both orientations(Fig. 3A). By use of this protocol, detectable levels of RMSSIintegration were measured in cells coinfected with AAV-GFPvectors and Ad-Rep in the presence of doxycycline but not inthe absence of Rep (Fig. 3B). Surprisingly, however, the use ofthe AAVp5D10-GFP vector did not significantly increaseRMSSI compared with results for the AAV-GFP virus (meanFIG. 3. Quantification of RMSSI in transduced HepaRG cells. (A) Human chromosome 19 region (GenBank accession no. AC010327) andposition of primers pAAVS1 (5-TCAGAGGACATCACGTG-3) and pITR (5-TTAACTACAAGGAACCCCTA-3) used for the amplificationof AAVS1-AAV junctions by quantitative PCR. D and A indicate the positions of the donor (5-TGTTGCTGCCCAAGGATGCT-FL; TIBMolbiol) and acceptor (5-LC Red640-TTTCCGGAGCACTTCCTTCTCG-p; TIB Molbiol) probes, respectively. The number of integrated AAVcopies in the AAVS1 locus was measured by using as a standard different copies of the pAAVS1-ITR control plasmid that carries an artificialAAVS1-chromosome 19 junction fragment (5, 6) and was further calibrated by using genomic DNA extracted from HA-16 cells that contain thewt AAV-2 genome integrated into the AAVS1 site in a head-to-tail configuration (2). Results are expressed as the percentage of AAVS1-ITRjunctions found in each sample relative to the number of junctions found using equivalent amounts of HA-16 cells. (B) Differentiated HepaRGcells were transduced with AAV-GFP or AAVp5D10-GFP in the presence or in the absence of a gutless adenovirus encoding Rep78 under thecontrol of a doxycycline-inducible promoter (12). Alternatively, the cells were infected with wt AAV-2 particles (multiplicity of infection, 4,000physical particles/cell and 20 infectious particles/cell for rAAV and adenoviral vectors, respectively). The percentage of RMSSI was measured forHepaRG cells 5 days after transduction. CTRL, genomic DNA from nontransduced HepaRG cells. (C) HeLa cells were transfected with theRep-expressing plasmid (pBGTetOCMVRepTS) and then infected with rAAV. Alternatively, the cells were infected with wt AAV2 particles. Bothinfections were performed at a multiplicity of infection of 4,000 particles/cell. RMSSI was measured by quantitative PCR 3 days after infection andculture in the presence of doxycycline.2592integration frequencies of 3.4  2.5% and 4.0  3.1%, respec-tively). To examine whether higher levels of site-specific inte-gration could be achieved using a viral genome with a p5element and the rep gene on the same DNA molecule, wemeasured RMSSI after infection of differentiated HepaRGcells with wt AAV-2 particles. The integration frequency usingwt AAV2 particles was approximately 0.5  0.3%, thus indi-cating that RMSSI in this cell type could not be enhanced evenusing this optimal substrate (Fig. 3B). Altogether, these resultsindicated that insertion of the minimal p5 element did notenhance RMSSI of a rAAV vector during infection of growth-arrested cells.To determine whether this result could be attributed to thecell type chosen for this assay, a similar experiment was con-ducted using HeLa cells that were transfected with the doxy-cycline-inducible Rep plasmid (pBGTetOCMVRepTS) andthen infected with AAV-GFP particles containing either thefull-length or the minimal p5 element. RMSSI was measuredby quantitative PCR for the cell population 3 days after infec-tion in the presence of doxycycline. By using this protocol, ahigher level of RMSSI was detected when HeLa cells wereinfected with wt AAV2 particles than with HepaRG cells (Fig.3C). When cells were infected with rAAV vectors, detectablelevels of RMSSI events were measured only in cells infected inthe presence of Rep. However, as previously observed forHepaRG cells, the insertion of the wt or minimal p5 elementin the rAAV vector did not significantly increase RMSSI com-pared with results for the AAV-GFP virus (mean integrationfrequencies of 23.3  13.5%, 29.5  13.2%, and 33.9  18.0%for AAV-GFP, AAVp5wt-GFP, and AAVp5D10-GFP, respec-tively).Altogether, this study documents for the first time the effectof the p5 element in the context of a viral infection. We showthat insertion of the p5 region, in either its wt or its minimalversion, cannot significantly increase RMSSI of a rAAV vectorduring infection of proliferating or quiescent human pseudo-primary cells. This result contrasts with the effect previouslyobserved in this and other studies analyzing RMSSI of p5-containing rAAV plasmids after transfection into individualcell clones. This differential effect may depend upon the rAAVsubstrate introduced into the cells (i.e., a plasmid versus alinear molecule) or the selection procedure performed to ob-tain individual cell clones. Further studies should be conductedto determine whether, likewise, insertion of the p5 element isdispensable for enhancing RMSSI of rAAV vectors in vivo.We thank Daniela Hu¨ser and Regina Heilbronn for providing adviceand materials to perform AAVS1 quantitative PCR, Christiane Guil-louzo-Guiguen for the HepaRG cells, and Nelly Robillard for fluores-cence-activated cell sorting.This work was supported by the Association Franc¸aise contre lesMyopathies (AFM), the Association Nantaise de The´rapie Ge´nique(ANTG), the Fondation pour la The´rapie Ge´nique en Pays de la Loire,and the European Commission (contract FP6-019038, Magselectofec-tion).REFERENCES1. Aninat, C., A. Piton, D. Glaise, T. Le Charpentier, S. Langouet, F. Morel, C.Guguen-Guillouzo, and A. Guillouzo. 2006. Expression of cytochromes P450,conjugating enzymes and nuclear receptors in human hepatoma HepaRGcells. Drug. Metab. Dispos. 34:75–83.2. Dutheil, N., F. Shi, T. Dupressoir, and R. M. Linden. 2000. Adeno-associ-ated virus site-specifically integrates into a muscle-specific DNA region.Proc. Natl. Acad. Sci. USA 97:4862–4866.3. Franc¸ois, A., M. Guilbaud, R. Awedikian, G. Chadeuf, P. Moullier, and A.Salvetti. 2005. The cellular TATA binding protein is required for Rep-dependent replication of a minimal adeno-associated virus type p5 element.J. Virol. 79:11082–11094.4. Gripon, P., S. Rumin, S. Urban, J. Le Seyec, D. Glaise, I. Cannie, C.Guyomard, J. Lucas, C. Trepo, and C. Guguen-Guillouzo. 2002. Infection ofa human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. USA99:15655–15660.5. Hu¨ser, D., and R. Heilbronn. 2003. Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equalkinetics and frequency. J. Gen. 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Virol. 74:11511–11521.VOL. 82, 2008 NOTES 2593

Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration

https://iris.unimore.it/bitstream/11380/612267/2/guilbaud%20et%20al.pdf

Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration.

Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration.

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Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia