Targeted Large DNA Insertion lower panel). Clones derived from HeLa cells with AAV Rep68-induced disruptions in the AAVS1 locus are indicated by solid arrows, whereas those that also have dHV.68/5939.F50 DNA inserted into this specific region of human chromosome 19 are discriminated by open arrows. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (C) Ten micrograms of total cellular DNA from 10 clones of DMD myoblasts co-infected with dHV.68/ 5939.F50 and hcAd.FLPe.F50 were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA species were subjected to Southern blot analysis with the AAVS1-specific probe (AAVS1 probe; upper panel) and, after its removal, with the two probes binding close to the termini of the dHV.68/5939.F50 genome (59 and 39 vector-specific probes; lower panel). Clones derived from DMD myoblasts with AAV Rep68-induced disruptions in the AAVS1 locus are indicated by solid arrows, whereas those that also have dHV.68/5939.F50 DNA inserted into this specific region of human chromosome 19 are discriminated by open arrows. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (D) Ten micrograms of total cellular DNA from 9 representative clones derived from HeLa cells infected with dHV.68/5939.F50 alone were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA fragments were subjected to Southern blot analysis with the AAVS1-specific probe. Marker, GeneRuler DNA Ladder Mix molecular weight marker. doi:10.1371/journal.pone.0003084.g006 integration into the AAVS1 locus and, consistent with the Southern blot data (Fig. 6 and Table 1), show that targeted integration events are more frequent in cells in which rep68 expression is up- regulated by FLPe than in cells in which the rep68 gene switch is not activated. AAV Rep78/68-dependent DNA integration into the AAVS1 locus is not site-specific in the strict sense since it does not take place at a single nucleotide position but within a relatively long stretch of DNA. As a result, AAVS1-exogenous DNA junctions generated through the AAV machinery are usually present as single copies within a population of stably transduced cells. Thus, the smear detected in Fig. 7B, lane 4 most likely reflects the amplification of a mixture of AAVS1-dHV.68.5939.F50 DNA junctions with various sizes as observed following wild-type AAV DNA integration. To investigate this, PCR products from the smear (Fig. 7B, lane 4) were inserted into the cloning vector pCR-Blunt II-TOPO. The PCR fragment corresponding to the DMD myoblast clone 3C5 (Fig. 7B, lane 2) was cloned in parallel. Plasmid DNA purified from randomly selected colonies was digested with EcoRI to release the inserts. As expected, agarose gel electrophoresis revealed differently sized inserts reflecting the variation in vector DNA insertion sites within the AAVS1 locus (data not shown). Finally, DNA sequencing of these inserts representing individual integration events revealed the precise nucleotide location at which target site and vector DNA covalently joined each other. Two examples are depicted in Fig. 7C. Altogether, these experiments complement the Southern blot data presented in Fig. 6 and firmly establish the capacity of rep68- positive dHV particles to accomplish insertion of large DNA at a specific locus in the human genome. Discussion The ability of wild-type AAV to insert its DNA into a specific genomic region in cultured cells of higher primates is unique among all known eukaryotic viruses. However, the AAV DNA integration mechanism remains sketchy, partially owing to its complexity [34]. For example, it is not known whether the pre- integration complexes consist of DNA molecules with linear, circular or both types of topologies. There is evidence, nonetheless, for genomic insertion through circular intermediates from studies showing (i) the capacity of rAAV plasmids to integrate into the AAVS1 locus, (ii) the generation of circular double-stranded DNA via AAV ITR-mediated intermolecular recombination and (iii) the presence in cells latently infected with wild-type AAV of head-to- tail proviral tandem repeats [34]. The unique structure of dHV genomes consisting of linear DNA molecules that lack AAV ITRs at one or both ends in their monomeric and dimeric configuration, respectively, precludes the generation of circular integration substrates via AAV ITR-mediated recombination. Our finding that Rep68 can catalyze the insertion of dHV DNA into the AAVS1 region thus suggests that linear templates are also amenable to the AAV Rep78/68-dependent integration process. Although detailed knowledge about the AAV DNA integration mechanism is lacking, researchers are actively pursuing the development of gene delivery systems that exploit the desirable feature of locus-specific integration provided by AAV elements [4,5,35,36]. When compared to non-targeted integrating vectors, these gene transfer systems might impose a lower risk of insertional oncogenesis caused by the inactivation of tumour suppressor genes or, as observed in clinical trials using gammaretroviral vectors, by the dysregulation of proto-oncogenes [37]. Among the many viral and non-viral vector systems capitalizing on the AAV integration machinery are those based on hcAd vectors [14,38,39]. Recchia and colleagues incorporated rAAV DNA encoding GFP and hygromycin B phosphotransferase into an hcAd vector genome [38]. Co-infection of Hep3B cells with the resulting vector and another hcAd vector containing a bacteriophage T7 promoter- driven rep78 gene at a multiplicity of infection (MOI) of 10 transducing units per cell each followed by hygromycin B selection revealed that the overall STF was 0.4% (i.e. 400 hygromycin B- resistant clones/105 target cells). According to the authors, similar results were obtained in other human hepatic cell lines [38]. Of note, the STF was not enhanced by AAV rep78 expression. However, Rep proteins did promote the insertion of the AAV Table 1. Frequencies of targeted foreign DNA integration events derived from Southern blot analysis of dHV.68/5939.F50- transduced HeLa cell and DMD myoblast clones. Randomly selected eGFP+ clones (N) Clones with AAVS1 disruptions (D)/N [%] Targeted integration events/N [%] Targeted integration events/D [%] doi:10.1371/journal.pone.0003084.t001 HeLa cells dHV.68/5939.F502 hcAd.FLPe.F50 40 0/40 [0] 0/40 [0] 0/0 [0] dHV.68/5939.F50+ hcAd.FLPe.F50 38 19/38 [50] 9/38 [24] 9/19 [47] DMD myoblasts dHV.68/5939.F50+ hcAd.FLPe.F50 18 5/18 [28] 5/18 [28] 5/5 [100] PLoS ONE | www.plosone.org 9 August 2008 | Volume 3 | Issue 8 | e3084