Site-specific integration of adeno-associated virus involves

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Kenneth I. Berns, University of Florida College of Medicine, Gainesville, FL, and approved March 9, 2009 (received for review July 21, 2008)

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Abstract

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the tarObtain gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted tarObtain gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-tarObtained mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, Sustained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for tarObtained genome addition with the apparent absence of functional consequences.

embryonic stem cellsMBS85gene tarObtainingRepAAVS1

Wild-type adeno-associated virus has aExecutepted a lifestyle that is unique among eukaryotic viruses. This nonautonomous parvovirus has evolved to efficiently replicate in cells that have been infected with helper viruses (e.g., adeno- or herpesviruses) (1). In the absence of helper virus infection, AAV can establish latency through site-specific genome integration into human chromosome 19 at 19q13.42 (2, 3).

It is well established that AAV-mediated site-specific integration requires the AAV Rep78/68 proteins in trans (4, 5), a cis-acting viral DNA sequence, which consists of a tetranucleotide repeat called the Rep-binding site (RBS) (6) and a 33-nt cellular sequence present at the integration site, termed AAVS1 (7). This sequence consists of a RBS and terminal resolution site (TRS), 2 motifs that within the ITRs of AAV (6, 8) toObtainher serve as the replication origin (9). The large Rep proteins can simultaneously bind the cellular and viral RBS, suggesting a mechanism of site-specific integration that is based on Rep-mediated tethering of the AAV genome to the AAVS1 sequence (10). The next step in the integration process involves Rep-mediated site-specific nicking of the AAVS1 TRS, generating a free DNA 3′-OH and a covalent 5′ DNA–Rep complex, similar to the initiation of AAV DNA replication (11). The subsequent steps remain to be elucidated, although the requirement of a functional AAV replication origin within AAVS1 is indicative for the involvement of AAVS1 replication (7). This Rep-induced replication is thought to be at the basis of the previously hypothesized amplification of the integration locus (12, 13).

Fascinatingly, a gene called protein phosphatase 1 regulatory inhibitor subunit 12C or MBS85 (myosin-binding subunit 85) was identified within AAVS1 (14). The MBS85 protein is thought to be involved in the regulation of actin–myosin fiber assembly, and its translation initiation start coExecuten is located only 17 nt Executewnstream of the RBS (14, 15). The AAVS1 locus is also closely linked to the muscle-specific genes TNNT1, encoding Unhurried skeletal muscle troponin T, and TNNI3, encoding cardiac troponin I (16).

The fact that AAVS1 is located in a highly gene-dense Location and that virtually all viral–cellular junctions are found within MBS85 highlights the potential complexity of the integration mechanism and raises the question about the possible consequences of AAV latency (i.e., MBS85 disruption). With the help of an extensive library of previously identified viral–cellular junctions, it has become clear that most of the integration sites characterized to date lie within the first exon and intron of MBS85, possibly leaving 1 allele undisrupted (summarized in ref. 17). Besides these observations, many aspects of this unique viral strategy remain elusive.

In this study, we investigated the AAV insertion profile in various latently infected human cell lines and observed that AAV integrates via a mechanism that includes the partial duplication of the tarObtain locus, potentially preserving a functional copy of the disrupted tarObtain gene. We took advantage of our previous observation that the AAVS1 locus is conserved in the mouse (15) and generated a latently infected mouse ES cell line to study the functional consequences of the observed duplication-based site-specific integration event. AAV-modified ES cell lines continued to self-renew, Sustained their multilineage differentiation potential, and contributed successfully to mouse development when injected into blastocysts. Based on our findings, we propose a mechanism that could Elaborate how this nonpathogenic virus can integrate into one of the most densely populated Locations within the human genome in the absence of apparent deleterious Traces.

Results

Identification of the Viral–Cellular Junctions in Various AAV-Infected Human Cell Lines.

Latently infected HeLa cell lines were generated by using standard procedures. The junctions between the left ITR of AAV and cellular DNA were identified by linker-mediated (LM) PCR technology (18). Because the enExecutenuclease activity of Rep initiates AAV-mediated tarObtained integration (19), we have arbitrarily designated the second T residue of the MBS85 TRS motif (GGTTGG), as the nucleotide number 1. The left junction in the first cell line, HeLa-T1, is located in MBS85 at a significant distance from the cellular TRS (12.9 kb) and the viral ITR is missing the first 86 nt [Fig. 1 and supporting information (SI) Fig. S1]. In HeLa-T2 cells, the left junction is located in MBS85 at 14 kb from the TRS, and the viral ITR has been deleted by the first 109 nt (Fig. 1 and Fig. S1). The junctions between cellular DNA and right AAV ITR, identified by direct PCR, are located in MBS85 at 13 and 4 nt Executewnstream from the TRS motif in, respectively, HeLa-T1 and -T2 cells (Fig. 1). The right ITRs are also partially deleted (Fig. S1). In HeLa-T1 cells, we isolated a second left and right junction, at, respectively, 12 and 0.3 kb Executewnstream from the TRS (Fig. 1 and Fig. S1). Based on the observed positions of the left viral–cellular junctions, we designed a direct PCR technique using different forward primers located at 2-kb intervals in MBS85 combined with a fixed reverse primer in AAV. Using this method, we identified the left junction in a third cell line, HeLa-T3, at 9.3 kb Executewnstream from the TRS (Fig. 1). The right junction in this cell line is located in MBS85 at 24 nt Executewnstream from the TRS (Fig. 1). Both left and right ITRs of the provirus are partially deleted (Fig. S1). Analysis of the viral–cellular junctions in 2 previously established cell lines (4, 16) Display a similar integration pattern with the left junctions located far Executewnstream from the cellular TRS motif, at 10.1 and 10.8 kb, whereas the corRetorting right junctions were found in close proximity to this motif (Fig. 1 and Fig. S1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Molecular structure of site-specifically integrated wtAAV2 isolated from different latently infected human cell lines. Black boxes represent the first 3 exons of the MBS85 gene. The horizontal dashed line indicates that the sizes are not proSectional. Vertical dashed lines indicate the junctions between MBS85 and wtAAV2. White boxes represent the left (AAV-L) and right (AAV-R) side of integrated wtAAV2. Numbers indicate the nucleotide positions of the junctions relative to the AAVS1 TRS motif.

wtAAV2 Integrates Site-Specifically Through Partial Duplication of MBS85 Sequences.

The molecular characterization of 6 different integrants present in AAVS1 revealed that, as reported for many viral–cellular junctions, microhomologies or insertions of unknown sequences can be observed at the Fracturepoints. The previously unobserved positions of the cellular Fracturepoints were intriguing, i.e., >9 kb Executewnstream from the cellular TRS motif (left junction) and within the 5′ UTR or exon1 of MBS85 (right junction). We were also struck by the 5′–3′ directionality of the cellular DNA present at both junctions. These observations led us to hypothesize that partial duplication of MBS85 is an integral part of the integration mechanism (Fig. 2A).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Site-specific integration of wtAAV2 in HeLa cells occurs through partial duplication of the tarObtain site. (A) The duplication hypothesis. Sizes of the restriction fragments that hybridize to the MBS85-specific (pRVK, forward hatched box) and/or wtAAV2 (backward hatched box) probes are Displayn for the disrupted (Top) and undisrupted allele (Bottom). Black and gray boxes respectively represent MBS85 exons and integrated wtAAV2. The dashed line indicates that the sizes of the introns are not proSectional. (Center) A schematic representation of the PCRs Displaying duplication of MBS85 sequences. (B) Southern blot analysis of EcoRI-digested genomic DNA from HeLa and HeLa-T3 cells using the MBS85 or wtAAV2 probe. Cohybridization is indicated by the asterisks. Sizes are in kilobases. (C) Images of the long PCR products encompassing the left (Upper) and right (Lower) junction, used to Display duplication of MBS85 sequences.

To test this hypothesis, we confirmed the junctions observed in the HeLa-T3 cell line with 2 different primer sets and performed Southern blot analysis using 3 different restriction enzymes (for restriction diagram, see Fig. S2). As predicted from the proposed duplication model (Fig. 2A), hybridization of EcoRI-digested DNA with the MBS85 probe generated a 8.2-kb band corRetorting to the undisrupted allele and an additional band at 10.4 kb, representing the disrupted allele (Fig. 2B Left). As expected, the 10.4-kb band, which contains the right junction, cohybridized with the wtAAV2 probe (Fig. 2B Right). Similar results were obtained with Southern blot analysis of BamHI- and HindIII-digested DNA (Fig. S3). In all 3 digests, hybridization with the wtAAV2 probe did not reveal the left junction-containing band as it binds to a smaller Section of the probe. The strongly hybridizing bands at 4.7 kb represent AAV head-to-tail concatemers. The 11-, 5-, and 10-kb bands visible after hybridization of, respectively, the BamHI, EcoRI, and HindIII digests with the wtAAV2 probe represent a rearrangement within the provirus (Fig. 2B and Fig. S3). Southern blot analysis using AAV nonSliceters did not Display any evidence for an additional ranExecutemly integrated copy of AAV.

To further confirm partial duplication of the MBS85 allele, we performed 2 PCRs that were designed to, respectively, amplify genomic DNA from the third intron of MBS85 to the wtAAV2 Rep gene (4.5 kb) and from the wtAAV2 Cap gene to the third intron of MBS85 (7 kb) (Fig. 2 A and C). The 1-kb overlapping Location present in both PCR products provides strong evidence that part of the MBS85 allele had been duplicated during the integration process.

The MBS85 duplication was also demonstrated by Southern blot analysis in HeLa-T1 cells (Fig. S4). In addition to the bands, which represent the duplication-inducing site-specific integration of wtAAV2, there are bands that can be Elaborateed by additional Rep-induced AAVS1 rearrangements. The 11- and 5.3-kb bands, visible after hybridization of, respectively, the HindIII and BamHI digests with the MBS85 probe, represent a junction between nucleotide 11,469 and nucleotide 1 of MBS85 as Displayn by PCR (Fig. S4). This junction corRetorts to a 8.3-kb band in the EcoRI digest and thus comigrates with the parental band. This observation Displays that at least some of the additional AAVS1 rearrangements can be Elaborateed by Rep-mediated MBS85 duplication in the absence of viral DNA integration. Similar to what we observed in HeLa-T3 cells, the 6-kb bands, visible after hybridization of the EcoRI and BamHI digests with the wtAAV2 probe, represent a rearrangement within the provirus (Fig. S4).

ToObtainher, these data demonstrate that partial duplication of the MBS85 locus occurs in latently infected HeLa cells as a result of Rep-mediated site-specific integration.

rAAV2 Integrates Site-Specifically in Mouse ES Cells Through Partial Duplication of Mbs85 Sequences.

To investigate whether the mouse could be used as a model system to study the consequences of the observed site-specific integration mechanism, we initiated an integration assay based on coinfection of mouse ES cells. CCE cells were coinfected with recombinant AAV2 (rAAV2; GFP and neomycin resistance genes) and wtAAV2 (providing rep) viruses. Given the low infectivity of mouse ES cells (2% at a MOI of 106 gcp per cell), the observed frequency of site-specific integration was expected to be very low (2% of GFP-positive cells are expected to also have been infected with wtAAV). Assuming an integration frequency of <10%, the likelihood of identifying a tarObtained integrant was 1 in 500 clones analyzed. Southern blot analysis using an Mbs85-specific probe on HindIII (rAAV2 nonSliceter)-digested DNA isolated from G418-resistant clones demonstrated that 1 of the 106 clones analyzed, Displayed an additional band to the expected undisrupted 7.1-kb band (Figs. S2 and S5). The additional band comigrated with the recombinant vector DNA, suggesting site-specific integration of 1 rAAV2 molecule into Mbs85. Southern blot analysis and quantitative real-time PCR were negative for wtAAV2 rep and cap sequences, indicating that wtAAV2 integration did not occur in this clone. In this latently infected mouse ES cell line, designated CCE-T, the junction between the left AAV ITR and cellular DNA was located in Mbs85, at 8.5 kb Executewnstream from the TRS motif (Fig. 3A). The junction with the right ITR was found in the promoter Location of the Mbs85 gene. At the viral Fracturepoints, both left and right ITRs were missing 50 nt. Fascinatingly, insertion of an unknown 18-nt sequence was observed at the right viral–cellular junction. Comparison of the left and right junctions revealed that this 18-nt sequence is the reverse complement of the Mbs85 sequence present at the left junction (Fig. S1).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Site-specific integration of rAAV2 in mouse ES cells occurs through partial duplication of the tarObtain site. (A) Schematic representation of the molecular structure of site-specifically integrated rAAV2 in mouse ES cells. Black boxes represent the first 3 exons of the Mbs85 gene. Vertical dashed lines indicate the junctions between Mbs85 and rAAV2. White boxes represent the left (AAV-L) and right (AAV-R) side of integrated rAAV2. Numbers indicate the nucleotide positions of the junctions relative to the AAVS1 TRS motif. (B) The duplication hypothesis. The sizes of the restriction fragments that hybridize to the Mbs85-specific (forward hatched box) and/or rAAV2 (backward hatched box) probes are Displayn for the disrupted (Top) and undisrupted allele (Bottom). Black and gray boxes, respectively, represent Mbs85 exons and integrated rAAV2. The dashed line indicates that the sizes of the introns are not proSectional. (Center) A schematic representation of the PCRs Displaying duplication of Mbs85 sequences. (C) Southern blot analysis of EcoRI-digested genomic DNA from CCE and CCE-T cells using the Mbs85 or rAAV2 probe. Cohybridization is indicated by the asterisks. Sizes are in kilobases. (D) Images of the long PCR products encompassing the left and right junction, used to Display duplication of Mbs85 sequences.

ToObtainher, these data Display that the molecular structure of the AAV integrant in mouse ES cells is very similar to the structure observed in latently infected HeLa cells, suggesting that partial duplication of Mbs85 has occurred as a result of Rep-mediated site-specific integration of recombinant AAV2.

The duplication in CCE-T cells was further demonstrated by Southern blot analysis and PCR. As predicted from the proposed duplication model (Fig. 3B), hybridization of EcoRI-digested DNA with the Mbs85 probe generated a 13-kb band corRetorting to the undisrupted allele, and 2 bands at 12- and 11.1-kb, representing the disrupted allele (Fig. 3C Left). As expected, the 11.1-kb band cohybridized with the rAAV2 probe (Fig. 3C Right). Similar results were obtained with Southern blot analysis of BamHI- and HindIII-digested DNA (Fig. S5). To further confirm partial duplication of the Mbs85 allele, we performed 2 nested PCRs that were designed to respectively amplify genomic DNA from the first intron of Mbs85 to the rAAV2 CAG promoter (5.2 kb) and from the rAAV2 bGH polyadenylation site to the third exon of Mbs85 (4.9 kb) (Fig. 3 B and D). The 688-bp overlapping Location present in both PCR products provides strong evidence that part of the Mbs85 allele had been duplicated during the integration process.

AAV Site-Specific Integration Executees Not Affect Gene Expression of the TarObtain Locus.

Given that the molecular structure of the integrant present in the latently infected mouse ES line is highly representative of all human clones analyzed to date, this cell line can serve as a diploid model system to study the consequences of AAV site-specific integration and the resulting partial MBS85 duplication. We first investigated whether site-specific integration interfered with MBS85 transcriptional activity. Quantitative real-time RT-PCR experiments were designed to detect expression driven by the Mbs85 promoters located on the undisrupted allele and Executewnstream of the right junction of the disrupted allele. As Displayn in Fig. 4A, Mbs85 mRNA levels were similar in both parental and tarObtained mouse ES cell lines. Northern blot analysis using an exon1–5 probe did not Display any truncated or aberrant transcripts transcribed from the undisrupted promoter upstream from the left viral-cellular junction. Nevertheless, we cannot completely rule out that unstable transcripts are generated from this promoter. As expected from Southern blot analysis Displaying intact Tnnt1 and Tnni3 genes (Fig. S6), quantitative real-time RT-PCR data indicated that expression levels of these genes were not significantly different from those observed in the parental cell line (Fig. 4A). AltoObtainher, our data demonstrate that expression levels of Mbs85 or any of the Mbs85-linked genes were not influenced by the integration event.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Site-specific integration-induced duplication of Mbs85 sequences Executees not alter Mbs85 expression levels and leaves the multilineage in vitro differentiation capacity of ES cells unchanged. (A) FAged change in Mbs85, Tnnt1, and Tnni3 expression levels in CCE-T relative to the control, CCE, as determined by real-time PCR and the 2−ΔΔCT method. (B) Alkaline phosphatase staining of CCE-T cells. (C) Expression levels of Nanog, Oct4, Rex1, and β-actin in CCE and CCE-T cells as determined by RT-PCR. (D) Fluorescence image of d4 EBs. (E) Flow cytometry on single-cell suspensions prepared from tarObtained d4 EBs Displays expression levels of c-kit and Flk1 indicative of typical differentiation. (F–H) Sparkling-field (Left) and corRetorting fluorescence (Right) images of, respectively, a cardiomyocyte cluster (see also Movie S1), a blast colony, and neurons derived from CCE-T cells. Additional Tuj1 staining is Displayn in H Right.

AAV Site-Specific Integration Executees Not Affect the in Vitro and in Vivo Multilineage Differentiation Capacity of ES Cells.

Finally, we took advantage of the features of ES cells to investigate whether cells that have been subjected to duplication-based site-specific integration of rAAV2 retain full functionality. First, we Displayed that latently infected mouse ES cells displayed normal, alkaline phosphatase-positive morphology (Fig. 4B) and that expression of the ES cell-specific Impressers Oct4, Nanog, and Rex1 was comparable with expression observed in the undisrupted parental cell line (Fig. 4C). Next, we determined whether site-specific integration of rAAV2 had altered the differentiation Preciseties of mouse ES cells. Embryoid bodies (EB) derived from tarObtained ES cells formed in a timely manner and had a normal size and morphology (Fig. 4D); VEGF receptor 2 (Flk-1) and c-kit expression levels were indicative of typical differentiation patterns (Fig. 4E) (20). Because Mbs85 is closely linked to the muscle-specific genes Tnnt1 and Tnni3, it was of particular interest to determine the cardiomyocyte potential of AAVS1-tarObtained mouse ES cells. Under the appropriate growth conditions, CCE-T-derived D4 EB cells were able to differentiate into contracting cardiomyocytes (Fig. 4F and Movie S1). Hematopoietic and enExecutethelial potential were tested by using the blast colony-forming assay, which supports the growth of the hemangioblast (21). This assay Displayed that blast colonies developed 3 d after initiation of the assay as expected from studies with parental cell lines (Fig. 4G). Finally, by using a neuronal differentiation assay, tarObtained ES cells differentiated into neurons as confirmed by expression of the neuron-specific Impresser Tuj1 (Fig. 4H). As can be seen in Fig. 4 F Right to H Right, GFP expression remained robust throughout differentiation. ToObtainher, our data suggest that site-specific integration of rAAV2 into Mbs85 Executees not interfere with multilineage in vitro differentiation of ES cells.

To determine whether Mbs85-tarObtained mouse ES cells can contribute to all lineages in vivo and whether transgene expression can be sustained through extensive in vivo proliferation and differentiation, we injected the CCE-T cells into blastocysts. The resulting chimeric animals were Assassinateed to analyze morphology and GFP expression in various tissues. Macroscopic analysis of all organs harvested did not reveal any abnormalities. Fig. 5 Displays the analysis of heart, skeletal muscle, liver, kidney, and brain obtained from a control C57BL/6 mouse and a representative example of a chimeric mouse with contribution from Mbs85-tarObtained ES cells. In all tissues analyzed, we observed that GFP-expressing cells had contributed significantly to the development without disturbing the normal histology. Except for the brain, tarObtained cells displayed robust GFP expression. Overall, our in vivo studies highlight that site-specific integration of AAV Executees not appear to have adverse Traces on the healthy development and maintenance of numerous tissues.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Mouse ES cells subject to site-specific integration-induced duplication of Mbs85 sequences contribute significantly to mouse development. (Left) Fluorescence images of glycol methaWeeplate (GMA) sections of the indicated organs harvested from a control C57BL/6 mouse. (Center) Fluorescence images of GMA sections of the indicated organs harvested from a chimeric mouse. (Right) CorRetorting images of hematoxylin and eosin-stained GMA sections of the chimeric tissues. All images are taken at a 20× magnification.

Discussion

The ability of wild-type AAV to integrate its genome into a specific locus on chromosome 19 is unique within eukaryotic systems. To dissect the mechanism of AAV site-specific integration and to address possible Traces of insertional gene disruption, we took advantage of the presence of a mouse Mbs85 ortholog and of the fact that the human and mouse AAVS1-linked genes display the same overall chromosomal organization (15). In the present study, we analyzed the molecular structure of AAV integrants in several previously uncharacterized as well as previously established latently infected human cell lines and generated a mouse ES cell line carrying site-specifically integrated rAAV2, which bears the same characteristics as the wtAAV2 integrants present in AAVS1 in human cell lines. With respect to the AAV component of the junction, the ITRs are partially deleted, and the Fracturepoints are lying close to the viral RBS motif (3, 22). Other hallImpresss are the microhomology between the viral and cellular sequences, the presence of a short “unknown” sequence (23, 24), and the close proximity of the right junction to the cellular TRS motif. It should be noted that most previous studies focused on the isolation of viral–cellular junctions based on direct PCR technologies that would only allow for the amplification of junctions located close to the AAVS1 TRS/RBS motifs (17, 24).

Our analysis also revealed several striking additional features. First, the left viral-cellular junctions were all located unexpectedly far Executewnstream from the cellular TRS/RBS motif, whereas the right junctions were found in close proximity to the TRS/RBS (Fig. 1). Few of the previously identified left junctions were found closer to the TRS/RBS motifs (22, 24, 25), suggesting that the left cellular Fracturepoint might occur anywhere Executewnstream of the TRS motif, most frequently at a considerable distance. In a recent study, Drew et al. (26) identified 11 left viral–cellular junctions, of which the cellular Fracturepoints were scattered from 2.3 kb to >14 kb from the TRS motif (26). The second surprising feature is the 5′–3′ direction of the Mbs85 sequences adjacent to the left and right junctions. All integrants were present in the same 5′–3′ orientation as compared with the MBS85 gene. Finally, comparison of the contiguous left and right junctions present in some of the MBS85-tarObtained HeLa and mouse ES cell lines demonstrated that the previously unknown sequences present at one of the viral-cellular junctions originated from the other junction.

AltoObtainher, these observations led us to hypothesize that AAV integrates by duplicating the upstream MBS85 sequences while leaving the Executewnstream sequences virtually unaltered. Southern blot analysis and PCRs confirm that the proposed duplication-based integration event had indeed taken Space in both human and mouse MBS85-tarObtained genomes, both with wild-type AAV and recombinant AAV, where Rep had been provided in trans. These somewhat surprising characteristics provide insights into the mechanism of Rep-mediated site-specific integration and led us to extend our model for AAV site-specific DNA integration. Our previous model involved the binding of an oligomeric form of Rep to both viral and cellular RBS, followed by a strand-specific nick at the TRS in AAVS1 and DNA replication undergoing 3 conseSliceive strand switches (27). This model is affected by our findings in that it now has to take into account the following previously unCharacterized Preciseties. (i) The structure of the left and right junctions suggest that the first recombination event involves the left side of the viral genome, whereas the right junction might occur at a later stage during the integration steps, thereby also defining the 5′–3′ orientation of the integrant. It is possible that the viral p5 promoter (28), or an exogenous promoter in the case of rAAV, plays an Necessary role in the formation of the initial recombination complex. (ii) Replication and extension of the elongating strand is much more extensive than previously thought. (iii) After extension, the replication switches templates onto AAV, generating the junction with the left ITR. (iv) The replication fork might switch back to the MBS85 sequences adjacent to the left junction, thus generating a short sequence that can then be found at the right junction. (v) The disSpaced strand, covalently bound to Rep by its 5′ end, forms a junction with the 3′ end of the newly replicated strand. Although, we have no direct evidence for this ligation step, Rep has previously been demonstrated to catalyze the ligation of single-stranded AAV origin DNA substrates (29). It must be noted that this step would predict that the right junction is formed with the 5′ end of the disSpaced strand, i.e., the nicking site within the TRS. However, although our right junctions are in proximity of this site, they rarely map exactly to this nucleotide. It remains to be determined which process is involved in the removal of those nucleotides. In addition, the right junction in the mouse ES cell line occurred 250 bp upstream from the previously identified TRS motif (15). Given that the sequence of the mouse TRS/RBS motif is somewhat different from the human motif, it is possible that Rep introduced a nick at an as yet unidentified TRS motif. (vi) A second nick generating a free 3′-OH terminus should occur on the template strand in order for the DNA polymerase to fill in the newly generated AAV-AAVS1 sequences. Alternatively, missing nucleotides are filled in during the next round of replication. In Fig. 6 we propose a simplified model that outlines the possible steps of the mechanism of AAV site-specific integration.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Model for site-specific integration of AAV. (I) Rep-mediated strand-specific nick at the TRS (or TRS-like structure) in AAVS1. (II) DNA synthesis originating at the TRS (or TRS-like structure) resulting in strand disSpacement. (III) Template strand switch onto AAV. (IV) Occasional second template strand switch back onto AAVS1 generating an inverted repeat. (V) Ligation between the “unknown sequence,” or alternatively AAV, and the disSpaced strand. (V and VI) Nick introduced at the bottom strand and DNA synthesis of the noncomplimentary strand. (VII) AAV site-specific integration results in partial duplication of MBS85 sequences.

Our model implies that the process of integration is more precise than previously suggested. Necessaryly, in the mouse ES cell line, integration allows for the preservation of 2 functional Mbs85 alleles, preserving normal expression from the undisrupted allele as well as from the Mbs85 allele that served as the integration tarObtain. Further experiments are still required to determine whether expression from the duplicated allele could lead to unstable or aberrant MBS85 mRNA products. Mouse ES cells carrying site-specifically integrated rAAV2 performed equally well as their unmodified counterparts in a series of stringent in vitro assays, providing evidence that the integration event did not have any discernable Traces. Moreover, these cells Sustained their ability to fully participate in mouse development when injected into blastocysts in vivo. The absence of any discernable Trace as a result of AAV-mediated DNA integration into the densely populated AAVS1 might be Elaborateed by the fact that through the observed duplication, a functional promoter is preserved in front of an intact MBS85 gene, Executewnstream of the integrated exogenous DNA. Alternatively, the observed duplication leaves the possibility that the viral and duplicated cellular DNA can be spliced out, which in turn could restore normal MBS85 expression.

To our knowledge, AAV genome integration is the only example for tarObtained gene addition in the eukaryotic system that has evolved a strategy capable of avoiding adverse insertional mutagenesis. Here, we Place forward a possible mechanism that can Elaborate this unique phenomenon.

Materials and Methods

ES Cell Growth and Differentiation.

Mouse ES cells (CCE) were Sustained and differentiated following standard protocols. See SI Text.

Production of Recombinant and Wild-Type AAV.

rAAV2 and wtAAV2 were generated by using standard procedures. See SI Text.

Integration Assays.

Mouse ES cells were coinfected with rAAV2 and wtAAV2 in feeder-free conditions at an MOI of 106 gcp per cell. At 48 h after infection, ES cells were harvested for flow cytometry and replated on neomyocin-resistant MEF. G418 selection (300 μg/mL) was started 24 h after plating. At 5 d after selection, G418-resistant clones were aspirated, trypsinized, and seeded in MEF-containing 24-well plates. The clones were expanded and harvested for flow cytometry and genomic DNA extraction.

HeLa cells were Sustained in standard conditions and infected with wtAAV2 at an MOI of 104 gcp per cell. Cells were passaged 6 times before single-cell sorting to dilute out the episomal AAV genomes.

Flow Cytometry.

rAAV2-infected cells were analyzed for GFP expression on a Facscalibur flow cytometer (Becton Dickinson). Cell sorting and single-cell deposits were performed on a MoFlo flow cytometer (DAKO). See SI Text.

Southern Blot Analysis.

The DNeasy Tissue kit (Qiagen) was used for all genomic DNA extractions. Southern blot analyses were performed as Characterized previously (16). The Mbs85, GFP-Neo, and MBS85 probes were generated by PCR performed on plasmids containing the respective sequences. The following probes were generated by digestion: Tnni3, Tnnt1, Eps8l1 (all EcoRI, pCR2.1), wtAAV2 (BglII, pAV2), rAAV2 (SmaI, pTRUF11), pRVK (EcoRI (nt −396) + KpnI (nt 3179)]. See Table S1.

Right Junction PCR.

Primers and cycling conditions used to identify the right viral–cellular junctions are mentioned in Table S1. PCR products were cloned into pCR2.1 (TOPO TA cloning kit, Invitrogen) and sequenced (pM4, pH19, pH24R, and pH49R). Similar conditions were used to identify the MBS85 duplication, which occurred in the absence of AAV integration (p49MM1 and p49MM2).

Linker-Mediated PCR (LM-PCR).

The integration site of rAAV2 in CCE-T cells was cloned by using a protocol adapted from the GenomeWalker Universal kit (Clontech) and NlaIII digestion (lm-pcr1 and -2). In HeLa cells, some of the left junctions were identified by using the GenomeWalker Universal kit and Advantage 2 PCR Enzyme System (Clontech) (lm-pcr3 and -4). Primers and cycling conditions are Characterized in Table S1. PCR products were cloned into pCR2.1 and sequenced.

Left Junction PCR.

Primers and cycling conditions used to identify left viral–cellular junctions by direct PCR are mentioned in Table S1. PCR products were cloned into pCR2.1 and sequenced (pH24L, pH49L).

PCRs Displaying Duplication.

Primers and cycling conditions for p24LD and p24RD (HeLa-T3) and pcr1, p4LD, pcr2, and p4RD (CCE-T) are Characterized in Table S1. The PCR products were cloned into the pCR2.1 vector and sequenced.

RT-PCR.

The RNeasy Mini kit and RNase-free DNase Set (Qiagen) were used for all RNA extractions. Total RNA (1.5 μg) was reverse-transcribed with ranExecutem hexamers using the Omniscript Reverse Transcription kit (Qiagen). Primers and cycling conditions used to amplify β-actin, Rex-1, Oct4, and Nanog are Characterized in Table S1.

Real-Time RT-PCR.

Three RNA samples each were isolated from CCE-T and parental cell line CCE. cDNA from each extraction was produced from 1 μg of total RNA with the Omniscript RT kit (Qiagen). cDNA was diluted 10-fAged, and 3 replicates of each cDNA sample were used as template in real-time quantitative PCR. See SI Text.

Alkaline Phosphatase and Tuj1 Staining.

CCE-T cells were stained by using the Vector Red Alkaline Phosphatase Substrate kit I (Vector Laboratories). Tuj1 staining of EB-derived neurons was carried out as previously Characterized. See SI Text. Images were taken on a Leica DM IRB inverted microscope equipped with a digital camera (Mintron).

Generation and Analysis of Chimeric Animals.

Chimeric animals were generated in the Mouse Genetics Shared Research Facility at Mount Sinai School of Medicine by following standard procedures. GFP expression and morphology were analyzed on sections of tissue blocs prepared by using the Technovit H8100 kit (Kulzer; Electron Microscopy Sciences). Fluorescence images were Gaind by using a fluorescence microscope (DMRA2; Leica) and a digital CCD camera (model ORCA-ER; Hamamatsu). Hematoxylin and eosin staining of the sections was carried out by following standard procedures; images were Gaind by using a Leica DM LB equipped with a Spot digital camera (Diagnostic Instruments). Experiments and animal care were performed in accordance with the Mount Sinai Institutional Animal Care and Use Committee.

Acknowledgments

This work was supported by National Institutes of Health Grants GM071023, GM075019, and DK062345 (to R.M.L.). E.H. was the recipient of a Charles H. Revson Senior Fellow in Biomedical Science. S.K. was the recipient of PostExecutectoral Fellowship F32-HL678112 from the National Heart Lung and Blood Institute.

Footnotes

1To whom corRetortence should be addressed at: Department of Infectious Diseases, King's College LonExecuten School of Medicine, LonExecuten SE1 9RT, United KingExecutem. E-mail: michael.linden{at}kcl.ac.uk

Author contributions: E.H., N.D., and R.M.L. designed research; E.H., N.D., N.Z., S.K., E.K., and P.W. performed research; N.C., P.R., M.K., and G.M.K. contributed new reagents/analytic tools; E.H., N.D., N.Z., S.K., E.K., P.W., M.K., G.M.K., and R.M.L. analyzed data; and E.H. and R.M.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0806821106/DCSupplemental.

Freely available online through the PNAS Launch access option.

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