Figure 1. Overview of the structure, production and applica- tion of dHVs. (A) Diagram of dHV shuttle plasmids. The structure of pdHV differs from that of pdHV.68/5939 and pdHV.68/3959 in that the former construct only codes for eGFP whereas the latter plasmids have, in addition, a FLP-inducible AAV rep68 expression module (see below) Targeted Large DNA Insertion inserted in between the Ad packaging signal and the AAV p5IEE in the 59R39 (pdHV.68/5939) or in the 39R59 orientation (pdHV.68/3959). Solid box, Ad ITR (hAd5 base pairs 1 through 103); Y, Ad packaging signal- containing sequence (hAd5 genome positions 104 through 454); shaded box with broken line, inducible AAV2 rep68 expression unit; shaded circle, p5IEE (AAV2 genome positions 153 through 291); line with subscript ‘‘dystrophin’’, stuffer DNA derived from human dystrophin cDNA; solid triangle, human growth hormone gene pA signal; open box, eGFP ORF; solid box with broken arrow, hEF1a promoter; shaded oval, AAV ITR (AAV2 nucleotides 1 through 145); open circle, prokaryotic origin of replication. (B) Schematic represen- tation of the rescue process of capsid-modified and rep68-positive dHV particles. Rescue of dHV DNA into hAd5 capsids containing chimeric fibers is initiated by releasing it from the plasmid backbone at the indicated PmeI restriction sites and transfecting the digestion products into the hAd5 E1-complementing PER.tTA.Cre76 cells. These cells are next infected with the E1-deleted helper Ad vector Ad.floxedY.F50, which supplies in trans all Ad DNA replication activities and Ad structural proteins (including chimeric fiber proteins) needed for the amplification and packaging of dHV DNA, respectively. Replication- and packaging-competent tail-to-tail dimers are assembled from input monomers due to Ad-dependent DNA replication and AAV ITR- mediated dimerization. The resulting dHV particles display on their surface hAd50 fiber domains and can be amplified by serial propagation in Ad.floxedY.F50-infected producer cells prior to purification in CsCl density gradients. The use of a helper Ad vector which has its packaging signal flanked by bacteriophage P1 loxP sites in combination with producer cells expressing the bacteriophage P1 cre gene strongly inhibits the formation of helper Ad particles. (C) Transduction pathway of rep68-positive dHVs. The FLP-inducible rep68 expression unit consists of a direct repeat of two FRT sequences flanking the AAV2 rep68 ORF followed by the herpes simplex virus thymidine kinase gene pA signal and the hGAPDH promoter. Since the transcription-promoting elements of the hGAPDH gene are positioned downstream of the Rep68-coding sequence, the rep68 ORF is not preceded by an active promoter and hence should not get expressed. To suppress rep68 activation by possible cryptic promoter elements located upstream of the rep68 initiation codon, a transcriptional terminator from the murine metallothionine gene was introduced immediately upstream of the 59 FRT site. The FLP-inducible rep68 expression module is located between the Ad cis-acting elements (i.e., the Ad ITR and the Ad packaging signal [Y]) and the AAV p5IEE. Excision and circularization of the FRT-flanked DNA by FLPe will position the hGAPDH promoter in front of the rep68 ORF and lead to the synthesis of Rep68. Finally, Rep68-mediated interactions between the RBEs in the vector DNA (i.e., AAV p5IEE and AAV ITR) and in human chromosome 19 (i.e. AAVS1 locus) will induce targeted chromosomal insertion of dHV DNA. doi:10.1371/journal.pone.0003084.g001 duplex dimers (DDs), respectively [6]. To activate the rep68-specific gene switch, we used hcAd.FLPe.F50. Negative and positive controls consisted of pGAPDH.Rep78/68- and pAAV.DsRed- transfected PER.tTA.Cre76 cells that were mock-infected and infected with Ad.floxedY.F50, respectively. Results depicted in Fig. 2 clearly show that accumulation of rAAV replicative intermediates in the pGS.pA+.Rep68-transfected PER.tTA.Cre76 cells is strongly stimulated by infection with hcAd.FLPe.F50 (i.e. in the presence of FLPe; compare lanes 1 and 2 of Fig. 2). In the absence of the recombinase only low amounts of rAAV replicative intermediates were generated (Fig. 2; lane 1). This suggests the existence of ‘‘leaky’’ rep68 expression at a level sufficient to allow limited amplification of rAAV DNA. Indeed, previous research has provided evidence that only few Rep78/68 molecules are required to induce AAV DNA replication [29]. The detection of rAAV- specific DMs and DDs in cells that received pAAV.DsRed, pGAPDH.Rep78/68 and Ad.floxedY.F50 (Fig. 2; lane 3) but not in cells containing exclusively pAAV.DsRed and pGAPDH.Rep78/ 68 (Fig. 2; lane 4) validated the assay. On the basis of these results, we conclude that pGS.pA+.Rep68 is FLPe-responsive and gives rise to the synthesis of functional Rep68 molecules. PLoS ONE | www.plosone.org 3 August 2008 | Volume 3 | Issue 8 | e3084