We have designed a novel HDA-EBV hybrid vector system capable of stably transforming mammalian cells with a high efficiency. Using coinfection of cells with two different HDA vectors, we were able to successfully deliver EBV episomes to different mammalian cell lines, as demonstrated by Southern blot and reporter gene assays. The HDA-EBV strategy resulted in acute transformation of about 40 to 50% of cells after 7 days and was therefore significantly more effective than EBV episome delivery by transfection of HDA plasmid DNA (≈5%). These data demonstrate that HDA-mediated delivery of EBV episomes is highly efficient compared to other methods. Furthermore, the binary nature of our system allowed manipulation of the doses for both the Cre-expressing and the target vectors and therefore optimization of transformation efficiency. As might be expected, the acute transformation efficiency increased significantly at higher target vector doses. However, higher total doses of HDA vectors also resulted in increasing cytopathic effects, possibly due to the exposure of cells to large amounts of adenovirus structural proteins. Interestingly, increasing the ratio of the Cre-expressing vector to the target vector resulted in a decrease in the acute transformation efficiency, likely due to competition between the two vectors during the multistep process of infection and recombination. The dose of vector necessary to generate a maximum number of episomes in a given cell population might depend on the cell type and the infection conditions.
Using the HDA-EBV hybrid system, we were able to generate a high percentage of stable puromycin-resistant transformants. The transformation efficiency of 43% was significantly greater than that (maximum of 10%) of a previously described first-generation adenovirus-EBV hybrid system (34
). The greatly enhanced efficiency of the HDA-EBV hybrid system is primarily a function of decreased cytotoxicity compared to that of the ΔE1 system, particularly at higher MOIs. Puromycin-resistant D17 transformants cultured in selective medium were able to maintain EBV episomes over 30 cell doublings, as evidenced by the persistent ability to form colonies in puromycin-containing medium and by detection of the full-length, unintegrated episome by Southern blotting (Fig. ). In the absence of selective pressure, the ability of D17 transformants to generate colonies in puromycin-containing selection medium as an indicator of the presence of episomal transgene expression decreased over time. The maintenance rate for D17 transformants was found to be 91% per cell division and was consistent with observations from previous studies indicating that the loss rate for EBV episomes was about 2 to 5% per generation in cells selected to be drug resistant following plasmid DNA transfection (25
It was somewhat surprising that the maintenance of EBV episomes was not dependent on the HDA target vector MOI. Cells initially infected at higher MOIs for HDA.PAC+FR were expected to contain a larger number of EBV episomes per cell and therefore to maintain puromycin resistance longer than cells infected at lower MOIs. It is possible, however, that the number of recombination products per cell reaches a maximum that is dependent on the level of Cre expression or the availability of target vector genomes inside the nucleus. Furthermore, cells may only be able to maintain a certain limited number of functional EBV episomes by means of chromosomal attachment mechanisms. Other EBV episomes that are not tethered to metaphase chromosomes may be lost at much higher rates during cell division.
Transgene expression in proliferating cells cultured under conditions that did not select for the presence of episomes revealed a biphasic loss of EBV episomes. After transformation of D17 cells by coinfection with HDA.CFP+FR and HDA.Cre, an initially rapid decline in the fractions of cells expressing CFP was followed by decline at a much lower rate (Fig. ). About 2 to 4% of cells continued to express CFP from an EBV episome at the end of 3 weeks. These findings are consistent with observations from previous studies in which plasmid DNA transfection was used to introduce EBV episomes (23
). Leight and Sugden (23
) estimated that the percentage of initially transfected cells able to maintain EBV episomes in a high percentage of daughter cells is between 1 and 10% under nonselective conditions. They suggested that the ability of cells to retain EBV episomes is dependent on epigenetic factors present in only a small fraction of cells in a given cell population. Even though EBV episomes were initially lost from dividing cells at a rapid rate, at least one daughter cell from every initially transformed cell must have retained the episome. Such cells remained puromycin resistant, explaining why the fraction of cells that were coinfected with HDA.PAC+FR and HDA.Cre and that gave rise to a drug-resistant colony (Fig. ) were similar to the fraction of cells initially expressing CFP after coinfection with HDA.CFP+FR and HDA.Cre (Fig. ). These cells remained puromycin resistant and gave rise to cells that segregated the plasmid to daughter cells at a high frequency, resulting in the formation of a drug-resistant colony. Once cells that segregate the episome to daughter cells arise, their descendants continue to segregate the episome at a high frequency, as shown by the low rate of episome loss from cells initially selected for growth in puromycin for 2 weeks (Fig. ).
The epigenetic mechanism underlying the efficient segregation of EBV episomes to daughter cells (23
) remains to be elucidated. However, the interaction of EBNA-1-bound episomes with cellular chromosomes required for episome segregation during mitosis likely involves cellular proteins in addition to EBNA-1. The cellular proteins p32/TAP and EBP2 have been reported to associate with the DNA-linking regions of EBNA-1 and may facilitate the interaction of EBNA-1 with mitotic chromosomes (4
). Hung et al. showed that a high-mobility group 1 or histone H1 protein sequence can substitute for EBNA-1 amino acids 1 to 378 and mediate the efficient accumulation of replicated oriP-containing plasmids, the association with mitotic chromosomes, nuclear retention, and long-term episome persistence (15
Extrachromosomally replicated and maintained EBV episomes have been studied for gene therapeutic applications by a number of investigators (3
). In cell cultures, an oriP- and EBNA-1-based construct was used to deliver the cystic fibrosis transmembrane conductance regulator gene to correct the cyclic AMP-dependent chloride transport defect in transformed human airway epithelial cells (22
). With an EBV episome, the stable expression of the human hypoxanthine phosphoribosyltransferase gene in a deficient lung fibroblast cell line was observed over 6 months (38
). EBV episomes have also shown promising results in animal models. A human factor IX-expressing EBV episome was used to transduce mouse liver and resulted in a 10- to 100-fold increase in transgene expression in the presence of EBNA-1 and the FR (33
). Furthermore, high levels of factor IX were found up to 8 months after injection. Lee et al. used an EBV episome for transfer of the human multidrug resistance (MDR-1) gene. In the context of EBNA-1 and oriP, transfected cells showed higher-level and more prolonged expression of MDR-1 in vitro and in vivo than did controls (21
The ability to deliver functional EBV episomes in vivo with our HDA-EBV system was confirmed after liver transduction of mice transgenic for albumin-Cre with a target vector. Transgene expression from the EBV episome was clearly dependent on the expression of Cre recombinase. In vivo, hepatocytes are mainly postmitotic, and liver tissue is therefore likely to maintain the EBV episome even in the absence of selection. It is also possible that only a small number of transduced cells are sufficient for therapeutic levels of transgene expression. Furthermore, EBV episomes may persist longer than linear HDA DNA because of resistance to exonucleases. An immune response to EBNA-1 is unlikely to present a problem, since the protein is poorly immunogenic (24
). However, countering an immune response against the products of potentially therapeutic transgenes may prove to be a formidable challenge. Future studies will assess the long-term persistence of EBV-mediated transgene expression in animals transgenic for albumin-Cre and in mice coinjected with HDA target vectors and HDA.Cre.
In summary, we have demonstrated the feasibility of delivering EBV episomes to mammalian cells in vitro and in vivo by using an HDA vector system. Further studies will evaluate the persistence of EBV episome-mediated transgene expression in vivo. Our system has the potential to significantly improve the duration and increase the level of transgene expression for therapeutic and diagnostic gene therapy applications.