and myoblasts) are refractory to transduction by conventional hcAd vectors, which are based on the species C human Ad (hAd) serotypes 2 and 5. This is explained by the virtual absence of the Ad and Coxsackie B virus receptor (CAR) on the surface of these cells [8– 11]. To allow efficient transduction of CAR-negative cell types, others and we have deployed a genetic retargeting strategy whereby cell receptor-interacting domains of the hAd serotype 5 (hAd5) fiber are substituted by those from other Ads resulting in hcAd particles pseudotyped with chimeric fibers [11–13]. In the present study, we describe the generation of dual hcAd/ AAV hybrid vectors (dHVs) [14] with modified capsids and with all the AAV cis- and trans-acting elements needed for locus-specific insertion of exogenous DNA. To avoid the well-described interference of the large AAV Rep proteins (i.e., Rep78 and Rep68) on the Ad-dependent DNA replication [15,16], which is required for vector genome amplification, we devised a FLP recombinase-dependent gene switch module to repress and activate rep68 expression in producer and target cells, respectively. Rep68 synthesis in target cells was induced with the aid of a fiber- modified hcAd vector encoding a thermostable version of the Saccharomyces cerevisae FLP recombinase (FLPe) [17,18]. Long-term cultures and clonal analyses of human cervix carcinoma (HeLa) cells and of myoblasts isolated from a patient with Duchenne muscular dystrophy (DMD) were used to determine stable transduction levels as well as the targeted chromosomal integration frequencies of the large DNA molecules delivered by capsid- modified and rep68-containing dHV particles. Activation of the inducible rep68 gene contained in the dHV genome resulted in increased stable transduction frequencies and was required for the efficient insertion of vector sequences into the AAVS1 locus. Moreover, Southern blot analyses of stably transduced HeLa cell and DMD myoblast clones suggest that locus-specific vector DNA integration occurs relatively more often in diploid than in genetically unstable target cells. Finally, our results suggest that linear double-stranded DNA molecules may serve as substrates for the locus-specific AAV integration machinery. Results Experimental strategy Since AAV Rep68 contains all the structural information needed to catalyze targeted DNA insertion, we decided to focus on the delivery into target cells of this instead of the larger Rep78 protein. However, because Ad-dependent DNA replication is strongly inhibited by the two large Rep proteins [15,16] rep68 expression during dHV production should be minimized. Indeed, generation of Ad-based vectors encoding Rep78 and/or Rep68 is not a trivial matter since even relatively low concentrations of these proteins in producer cells lead to the emergence and selection of mutant vectors with deletions in the rep sequences [19], and our unpublished results. Another point of concern is the stability of Ad vector genomes containing integration modules consisting of classic recombinant AAV (rAAV) genomes (i.e. ITR- transgene[s]-ITR). Investigations have shown that the presence of pairs of non-terminal inverted repeats in Ad vector genomes leads to replication-induced recombination through these sequences causing deletions/rearrangements in the vector DNA [20,21]. Ad vectors containing both rAAV genomes and rep78 and/or rep68 genes may, in addition, suffer from Rep78/68-mediated rescue (i.e. excision) of the rAAV DNA from a fraction of the vector backbones causing transgene loss. The origin of this problem lies in the fact that, although Rep synthesis can, to a great extent, be inhibited by using stringent inducible gene expression systems, most if not all of them are ‘‘leaky’’ (especially in the context of Targeted Large DNA Insertion replicating DNA templates). During the production of rep78/68- positive Ad vectors, producer cells will thus contain not only Ad- derived AAV helper activities (i.e. Ad gene products necessary for AAV DNA rescue and replication [6] but also Rep78/68 proteins. Finally, permanent expression of the rep78/68 gene at high levels is detrimental for many cell types causing cell cycle arrest and apoptosis [22–24]. Given all these potential pitfalls, we decided to construct hcAd- based vectors containing rep68 genes and Rep78/68-binding elements (RBEs) using a unique vector design in which FLP recombinase-dependent rep68 expression units are incorporated into the non-integrating parts of dHV genomes [14]. Previous results have shown that Ad-dependent DNA replication in concert with AAV ITR-mediated dimerization is the main mechanism responsible for the generation of dHV genomes from input monomeric templates molecularly cloned in shuttle plasmids [14]. These monomeric DNA molecules contain an Ad ITR, Ad packaging signal and AAV p5IEE at one end and an AAV ITR at the other end (Fig. 1A). The particular design of the resulting dHV genomes with AAV ITR sequences at the axis of symmetry and with the p5IEE at both extremities allows the introduction into target cells of DNA molecules structurally resembling wild-type AAV proviruses [25]. Resolution of the Holliday-like AAV ITR hairpin structures at the axis of symmetry either by cellular factors [14,26] or by Rep68-mediated nicking at the terminal resolution site (trs; Fig. 1B) will generate monomer-sized replicative intermediates that can be reutilized by the Ad replication machinery to produce new dHV genomes. Because they possess a favorable size for efficient packaging into Ad capsids (i.e. between 27 and 38 kb) [14,27] unresolved dimeric replicative intermediates will be preferentially packaged into Ad capsids [14] thus giving rise to dHV particles (Fig. 1B). The localization of the FLP-inducible rep68 gene upstream of the p5IEE in the rep68- positive dHV genomes (Fig. 1C) should impede its Rep68- dependent insertion into the AAVS1 locus [14]. To further reduce the likelihood of integrating rep68 sequences into the AAVS1 locus of target cells, the rep68 expression module was designed in such a way that its FLPe-mediated activation is accompanied by its physical separation from the gene(s)-of-interest. Circularization of the FLP recombinase target (FRT)-flanked DNA by FLPe positions a promoter in front of the rep68 open reading frame (ORF) resulting in Rep68 synthesis. Finally, Rep68-mediated interactions between the RBEs in the dHV genomes (i.e., AAV p5IEE and AAV ITR) and in human chromosome 19 (i.e. AAVS1 locus) should facilitate targeted insertion of the foreign DNA (Fig. 1C). Construction and testing of a FLP-dependent rep68 expression unit We started by constructing an inducible rep68 expression unit on the basis of the FLP/FRT site-specific recombination system of Saccharomyces cerevisiae. To this end, the coding sequence for DsRed.T4 in pGS.pA+.DsRed [28] was replaced by that of Rep68 giving rise to construct pGS.pA+.Rep68. The functionality of pGS.pA+.Rep68 was tested in a transient transfection assay. This assay consisted of introducing into pGS.pA+.Rep68-containing PER.tTA.Cre76 cells, AAV helper functions and an rAAV vector shuttle plasmid through infection with Ad.floxedY.F50 and transfection of pAAV.DsRed, respectively. In the presence of AAV helper functions plus functional AAV Rep68 molecules, rAAV genomes are expected to be rescued (i.e. excised) from plasmid backbones and to originate de novo synthesized replicative interme- diates consisting of double-stranded full-length monomeric and dimeric vector DNA, also known as duplex monomers (DMs) and PLoS ONE | www.plosone.org 2 August 2008 | Volume 3 | Issue 8 | e3084