clones containing stably transduced Mo7e cells. However, the percentage of GFP-positive cells in individual Mo7e clones varied greatly, which makes it very hard to accurately determine the actual STF. Nonetheless, from data presented in Fig. 9 of the study by Wang and Lieber [39] one can deduce that the STF at best was in the order of a few percent. Substitution of the rep78-positive for a rep78-negative hcAd vector resulted in somewhat lower stable transduction levels [39]. Finally, in 30% of the stably transduced clones derived from Mo7e cells co-infected with the rAAV- and rep78-containing hcAd vectors, the foreign DNA was found to be inserted into the AAVS1 region [39]. In this study, we report the generation of tropism-modified dHVs containing AAV rep68 expression units and show their ability to mediate integration of DNA fragments of at least 10.3 kb into the AAVS1 locus on human chromosome 19 in HeLa and muscle progenitor cells. Side-by-side rescue and propagation experiments revealed that the growth properties of the rep68- positive dHVs are very similar to that of the rep68-negative control vector. These results signify that the amounts of Rep68 made during the rescue and propagation of the rep68-positive dHVs were not enough to markedly inhibit their production. Transduction of rapidly proliferating human cells with dHV.68/5939.F50 particles followed by the activation of the rep68 gene switch incorporated in the vector genome led to the insertion at 19q13.3-qter of large DNA molecules that included a functional transgene expression unit and human dystrophin-coding sequences. Significantly, in HeLa cells exposed exclusively to dHV.68/5939.F50 (i.e. hcAd. FLPe.F50-negative cells), the basal Rep68 levels did not suffice to disrupt AAVS1 loci and, in agreement with this observation, no targeted DNA insertion could be detected in any of the 40 clones that were analyzed (Fig. 6). However, by using a more sensitive PCR-based assay, evidence for low-level AAVS1-targeted vector DNA insertion in FLPe-negative cells was found (Fig. 7B, lane 3). An interesting aspect of the present study is the side-by-side comparison of the performance of an AAV Rep78/68-dependent DNA targeting system in cell types with very different genetic stabilities. HeLa cells are notorious for their aneuploidy and widespread translocations, whereas the human myoblasts used in this study remain diploid even after many cell divisions [31]. If one considers only those cells in which Rep68 induced unambiguous AAVS1 disruptions, the targeted DNA insertion efficiency reached 47% in HeLa cells (9/19 clones) and a remarkable 100% in DMD myoblasts (5/5 clones). It is thus tempting to speculate that the lower targeted DNA integration proficiency of hybrid vector genomes in HeLa cells is a consequence of the higher genetic instability of these tumor cells causing a substantial fraction of vector genomes to insert randomly at double-stranded breaks possibly through non-homologous end-joining as observed with rep-negative rAAV DNA [41]. This would suggest that in the presence of appropriate amounts of Rep78 and/or Rep68 molecules, dHV DNA might indeed display a marked preference for locus-specific insertion in the genomes of therapeutically relevant (i.e. non-transformed) cell types. Further experiments that might include tests on a larger panel of cell types are warranted to establish or disprove this postulate. Importantly, we demonstrated that transduction of human cells with capsid-modified dHV particles can lead to long-term transgene expression in an AAV Rep68-dependent manner. However, notwithstanding the diverse experimental setups used, a common feature of this and other Rep78/68-dependent integrating hcAd vector-based gene carriers is their overall low STFs. The low STFs reached by these hybrid vector systems are at odds with those reported for wild-type AAV in human cells in culture. For instance, using Southern blot analyses, Hamilton and Targeted Large DNA Insertion colleagues deduced that wild-type AAV can stably transduce up to 50% of infected HeLa cells [42]. In addition, by deploying a quantitative real-time PCR assay, other investigators showed that wild-type AAV DNA integrates at 19q13.3-qter in at least 15% of infected HeLa cells [43,44]. Recchia and colleagues found for their monopartite hcAd/AAV hybrid vector system that the efficiency of AAVS1-specific integration was directly proportional to the Rep78 level in the target cells [40]. However, as mentioned previously, above a certain concentration, which might be different for different cell types, the two largest AAV Rep proteins have cytostatic or even cytotoxic properties. The low STFs achieved by the Rep78/68-dependent integrating hcAd vectors in long-term experiments deploying rapidly proliferating cells may thus relate to uncontrolled Rep78/68 synthesis. According to this hypothesis, many of the cells that have vector DNA integrated into the AAVS1 locus will display a strong growth disadvantage due to (persistent) high Rep78/68 levels. This will result in the selection of cells that contain no or few Rep78/68 molecules and are therefore not stably transduced or have the vector DNA integrated outside of the AAVS1 region. The idea of a strong selection against cells displaying high Rep68 activity is consistent with the observation that although most cells that initially received dHV.68/5939.F50 were also transduced by hcAd.FLPe.F50, a significant proportion of the randomly isolated HeLa cell and DMD myoblast clones did not show evidence of Rep68-induced AAVS1 disruptions (i.e., 50 and 72%, respectively). Future hcAd/AAV hybrid vector systems should therefore be designed in such a way as to ensure that the Rep78/68 activity is minimal in producer cells and high but short- lived in target cells. Inducible gene expression cassettes encoding Rep78 and/or Rep68 variants whose stability or subcellular localization can be controlled by cell-permeable small-molecule drugs may help to fulfill these requirements. Such controllable gene expression may, in addition, allow the production of rep68- positive dHV particles that are not dependent on the introduction of FLPe or on any other foreign protein into target cells. Materials and Methods Cells Culturing conditions for the human cervical carcinoma (HeLa) cells (American Type Culture Collection), the hAd5 early region 1 (E1)-deleted and bacteriophage P1 cre-expressing PER.tTA.Cre76 cells and the DMD myoblasts have been described elsewhere [11,31,45]. DNA constructions Recombinant DNA techniques were performed by using established methods [46] or following the instructions supplied with specific reagents. Large-scale plasmid DNA purifications were carried out using JetStar 2.0 kits (Genomed) according to the manufacturer’s recommendations. All DNA constructs as well as their nucleotide sequences are available upon request. Viral vectors The production and titration of stocks of the E1-deleted and capsid-modified helper Ad vector Ad.floxedY.F50 have been previously described [11]. Ad.floxedY.F50 particles display at their surface chimeric fiber proteins consisting of basal shaft sequences from hAd5 and apical shaft and knob domains from hAd50 [11]. The rescue and propagation of dHV.F50, dHV.68/ 3959.F50 and dHV.68/5939.F50 were carried out essentially as described elsewhere [11,14]. The titers of dHV.F50, dHV.68/ 3959.F50 and dHV.68/5939.F50 stocks were determined by flow PLoS ONE | www.plosone.org 11 August 2008 | Volume 3 | Issue 8 | e3084