Figure 2. Testing the functionality of pGS.pA+.Rep68 and hcAd.FLPe.F50. Extrachromosomal DNA was extracted from PER. tTA.Cre76 cells co-transfected with the rAAV vector shuttle plasmid pAAV.DsRed and the FLP-activatable rep68 expression plasmid pGS.pA+.Rep68 and infected 24 h later with Ad.floxedY.F50 alone (lane 1) or with Ad.floxedY.F50 plus hcAd.FLPe.F50 (lane 2). Positive (lane 3) and negative (lane 4) controls consisted of extrachromosomal DNA extracted from Ad.floxedY.F50- and mock-infected PER.tTA.Cre76 cells, respectively, co-transfected with pAAV.DsRed and the constitutive rep78/68 expression plasmid pGAPDH.Rep78/68. The extracts were treated with DpnI to selectively digest input, non-replicated, prokary- otic DNA and were subjected to Southern blot analysis using a probe corresponding to the DsRed.T4 ORF. Lane M, GeneRuler DNA Ladder Mix molecular weight marker (Fermentas). The positions and sizes (in kb) of the rAAV replicative intermediates (i.e., DMs and DDs) are indicated. The numerals at the left correspond to restriction DNA fragment sizes in kb. doi:10.1371/journal.pone.0003084.g002 Rescue, propagation and characterization of rep68- positive and fiber-modified dHV particles Next, the FLP-inducible rep68 expression unit from pGS.pA+.Rep68 was inserted in either orientation upstream of the p5IEE in the dHV shuttle construct pAd/AAV.EGFP [14], hereafter referred to as pdHV, to generate pdHV.68/5939 and pdHV.68/3959, respectively (Fig. 1A). pdHV.68/5939, pdHV.68/ 3959 and the rep68-negative control plasmid pdHV were digested with PmeI and individually transfected into PER.tTA.Cre76 cells. The PmeI digestion releases the Ad ITRs from almost the complete plasmid backbones turning them into much more efficient substrates for the initiation of Ad-dependent DNA replication than undigested templates. Subsequently, helper activities necessary for the amplification and packaging of dHV genomes were provided in trans by the infection of producer cells with the capsid-modified helper Ad vector Ad.floxedY.F50. After exhibiting complete cytopathic effect, the cell cultures were harvested and dHV seed stocks were prepared. These crude vector preparations were used to amplify dHV particles through four successive rounds of infection in Ad.floxedY.F50-infected Targeted Large DNA Insertion PER.tTA.Cre76 cells. Direct fluorescence microscopy revealed an increase in the frequency of enhanced green fluorescent protein (eGFP)-positive producer cells following each consecutive round of vector amplification (Fig. 3A). Quantification of functional vector particles by eGFP-directed flow cytometry showed that the propagation kinetics of the two different rep68-positive dHVs was very similar to that of their rep68-negative counterpart (Fig. 3B). Both rep68-negative and rep68-positive fiber-modified dHV particles obtained after four rounds of propagation were purified by isopycnic CsCl density gradient ultracentrifugation, titrated and used to transduce CD46-positive target cells that either do (HeLa cells) or do not (DMD myoblasts) express CAR at their surface. Direct fluorescence microcopy (Fig. 4A) and flow cytometry (Figs. 4B and 4C) showed a clear dose-dependent increase in gene transfer activity in both cell types. Moreover, flow cytometric quantification of the frequency of eGFP-positive cells (Fig. 4C, left graphs) and the average eGFP level per transduced cell (Fig. 4C, right graphs) revealed that, for each given vector dose, dHV.F50 and dHV.68/5939.F50 gave similar transduction levels. To investigate whether dHV.68/5939.F50 particles contain FLP-responsive rep68-specific gene switches, we performed Western blot analysis on lysates derived from HeLa cells transduced with CsCl density gradient-purified dHV.F50 and dHV.68/5939.F50 particles using a monoclonal antibody recog- nizing all four AAV rep gene products. HeLa cells were infected with dHV.F50 or dHV.68/5939.F50 alone (Fig. 5A; lanes 2 and 4) or in combination with hcAd.FLPe.F50 (Fig. 5A; lanes 1 and 3). Co-infection of the target cells with dHV.68/5939.F50 and hcAd.FLPe.F50 resulted in robust induction of rep68 expression (Fig. 5A; lane 3). In contrast, the amount of Rep68 produced in dHV.68/5939.F50-infected HeLa cells in the absence of FLPe was very low (Fig. 5A; lane 4). These results demonstrate dHV.68/ 5939.F50-mediated transfer of functional rep68 gene switch modules into target cells. Subsequently, to examine the synthesis of Rep molecules at the single-cell level we carried out eGFP direct fluorescence microscopy and Rep68 immunofluorescence micros- copy on HeLa cells co-transduced with dHV.F50 and hcAd.FL- Pe.F50 (Fig. 5B; left panels) or with dHV.68/5939.F50 and hcAd.FLPe.F50 (Fig. 5B; right panels). The cells infected with the rep68-negative dHV served to establish background levels for the Rep68 immunostainings. Despite the possibility that not all target cells were transduced simultaneously by both vector types, the overlay of the eGFP- and Rep68-specific signals (Fig. 5B; lower row) demonstrated co-synthesis of both proteins in most of the cells exposed to dHV.68/5939.F50 and hcAd.FLPe.F50. As expected, eGFP located in the cytoplasm and in the nucleus whereas Rep68 was present in the latter compartment. Moreover, generally, a direct correlation between the strength of the eGFP- and the Rep68-specific signals was observed. Permanent genetic modification of HeLa cells and myoblasts from a DMD patient by rep68-positive and fiber-modified dHV particles Finally, we tested the ability of dHV.68/5939.F50 to perma- nently transduce target cells by locus-specific transgene integration using two different human cell types, i.e., HeLa cells and DMD myoblasts. HeLa cells were chosen for this purpose because they constitute a reference cell line on which both wild-type AAV and Rep78/68-dependent DNA integration have been evaluated both in terms of stable transduction levels and targeted DNA insertion proficiencies. Myoblasts are being deployed in clinical trials for more than a decade to develop an allogeneic cell therapy for DMD [30]. One of the problems associated with this treatment modality is that it depends on strong and durable immunosup- PLoS ONE | www.plosone.org 4 August 2008 | Volume 3 | Issue 8 | e3084