The FLPfrt System for Generating HDAds

Based on the Cre/loxP strategy, systems were later developed using the yeast site-specific recombinase FLP, which catalyzes recombination between 34-bp frt sites (55). In this case, the packaging signal in the helper viral genome is flanked by frt sites so that, following infection of 293 cells stably expressing FLP, the packaging signal is excised, rendering the helper viral genome unpackagable but still able to replicate and trans-complement propagation of the coinfecting HDAd. This system was shown, by direct comparison, to be compara

Figure 2 The Cre/loxP system for generating HDAds. The HDAd contains only —500 bp of as-acting Ad sequences required for DNA replication (ITRs) and packaging (^), the remainder of the genome consists of the desired transgene, in this case, a LacZ expression cassette, and non-Ad ''stuffer'' sequences. The HDAd genome is constructed as a bacterial plasmid (pC4HSULacZ in this example) and is liberated by restriction enzyme digestion (e.g., PmeI). To rescue the HDAd (HDAdC4HSULacZ), the liberated genome is transfected into 293Cre cells and infected with a helper virus bearing a packaging signal flanked by loxP sites (e.g., AdLC8cluc,54). The helper viral genome also contains a stuffer sequence in E3 to prevent the formation of RCA in 293-derived cells. Cre-mediated excision of ^ renders the helper virus genome unpackagable, but still able to replicate and provide all the necessary trans-acting factors for propagation of the HDAd. The titer of the HDAd is increased by serial coinfections of 293Cre cells with the HDAd and the helper virus. Shown for the HDAd and the helper virus are the relevant Bgll sites and the corresponding DNA fragment sizes, as well as the location of the packaging signal probe (probe used for Southern blot hybridization analysis illustrated in Fig. 3.

Figure 2 The Cre/loxP system for generating HDAds. The HDAd contains only —500 bp of as-acting Ad sequences required for DNA replication (ITRs) and packaging (^), the remainder of the genome consists of the desired transgene, in this case, a LacZ expression cassette, and non-Ad ''stuffer'' sequences. The HDAd genome is constructed as a bacterial plasmid (pC4HSULacZ in this example) and is liberated by restriction enzyme digestion (e.g., PmeI). To rescue the HDAd (HDAdC4HSULacZ), the liberated genome is transfected into 293Cre cells and infected with a helper virus bearing a packaging signal flanked by loxP sites (e.g., AdLC8cluc,54). The helper viral genome also contains a stuffer sequence in E3 to prevent the formation of RCA in 293-derived cells. Cre-mediated excision of ^ renders the helper virus genome unpackagable, but still able to replicate and provide all the necessary trans-acting factors for propagation of the HDAd. The titer of the HDAd is increased by serial coinfections of 293Cre cells with the HDAd and the helper virus. Shown for the HDAd and the helper virus are the relevant Bgll sites and the corresponding DNA fragment sizes, as well as the location of the packaging signal probe (probe used for Southern blot hybridization analysis illustrated in Fig. 3.

ble to the Cre/loxP system in terms of efficiency of HDAd amplification and low helper virus contamination levels (55). A similar FLP-based system for generating HDAds was also reported by Umana et al. (59). The availability of 2 alternative systems for generating HDAd should expand the utility of HDAds by permitting use of recombinase-activated ''molecular switches'' (60) in which one recombinase can inhibit helper virus propagation while the other regulates transgene expression. Furthermore, it has been suggested that the 2 site-specific recombinase systems may be combined to further reduce the level of helper virus contamination, perhaps by flanking the packaging signal with both loxP and frt sites and using producer cells that express both Cre and FLP (55).

E. Characteristics of the Helper-dependent Vector

In addition to the minimal ds-acting Ad sequences required for DNA replication and encapsidation, Sandig et al. (56) showed that inclusion of a small segment of noncoding Ad sequence from the E4 region adjacent to the right ITR increases vector yields, possibly by enhancing packaging of the HDAd DNA. Early studies of Ad have established a maximum packaging capacity of 105% of the wild-type genome (~37.8 kb) (61). Subsequently, using the Cre/loxP system for generating HDAds, Parks et al. (47) established that the minimum genome size for efficient packaging into Ad virions was 27 kb.

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