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Serial Passage Number clinical trials (70). Like FG and multiply deleted Ads, the physical titer of HDAds can be readily obtained by measuring OD260. Measurements of infectious titers are, however, not as straightforward because, unlike FG and multiply deleted Ads, HDAds cannot form plaques on cell monolayers, thus precluding infectious titer determination by standard plaque assays. If the HDAd contains a transgene that can serve as a reporter, such as LacZ, an infectious titer can be obtained (see, for example, Fig. 3A). However, this is not the case with the majority of therapeutic HDAds, thus rendering the infectivity of HDAd preparations difficult to ascertain. Kreppel et al. (71) developed a DNA-based method of measuring the infectivity of HDAds that assays ability of the vector to deliver its genome into target cells. However, it is important to note that this method measures only cellular entry of the vector DNA, whereas infectious titer assays for FG and multiply deleted Ads also depend on viral gene expression (resulting in plaque formation or reporter gene product) following entry of the genome into the nucleus and are thus more stringent.

F. Characteristics of the Helper Virus

The most commonly used helper virus is a serotype 5, FGAd (E1 deleted) with its packaging signal flanked by loxP sites (Fig. 2). As with all FGAds propagated in 293 or 293-derived cells, the potential exists for the generation of replication-competent Ad (RCA; E1+ ) as a consequence of homologous recombination between the helper virus and the Ad sequences present in 293 cells. To prevent the formation of RCA, a ''stuffer'' sequence was inserted into the E3 region to render any E1+ recombinants too large to be packaged (54). The length of the E3 stuffer is such that the total size of the helper virus genome is <105% of wild-type, but >105% following homologous recombination with Ad sequences in the 293 cells. As of this writing, the emergence of RCA has yet to be reported using helper viruses with an E3 stuffer. In contrast, RCA is readily detected using helper viruses without an E3 stuffer when propagated in 293 and 293-derived cells (41,54). The choice of sequence used as stuffer in the E3 region may be important as it has been observed that some E3 inserts result in poor virus propagation, perhaps due to interference with fiber expression (Authors' unpublished data). Although it remains unclear what sequences constitute a good E3 stuffer, in general, noncoding sequences with no homology to the HDAd would be preferred. A number of sequences have been found to be stable and not to adversely affect virus propagation, including bacterial plasmid sequences, bacteriophage X sequences, and human DNA (54-56,59,72). The use of other E1-complementing cell lines engineered to preclude the formation of RCA is another option (73,74). Such cell lines have been used for the production of RCA-free FGAd, but there are not yet any reported of the development of Cre-expressing derivatives and their use for the production of HDAd.

One strategy to further enhance safety is to attenuate the helper virus to reduce its toxicity. To this end, a helper virus with a deletion of E2a was developed by Zhou et al. (75). E2a encodes the Ad 72-kD DNA-binding protein, which is essential for viral DNA replication. In theory, the E2a-deleted contaminating helper viruses would be unable to undergo viral DNA replication and thus would be unable to express the late Ad structural proteins, which are responsible for cytotoxicity and induction of cellular immunity. However, whether this modification truly improves safety is questionable, considering some studies have shown that the immune response and toxicity elicited against Ad is the same regardless of whether the vector has a defective E2a (21,41) (also see Section IV).

Because the HDAd genome does not integrate into the host chromosomes, but rather presumably remains episomal, it is likely that transgene expression will not be permanent. If transgene expression fades over time, it would be desirable to simply readminister the vector. Unfortunately, this simplistic approach is not possible because the initial administration elicits immunity in the form of neutralizing anti-Ad antibody, which renders subsequent readministrations ineffective. One strategy, known as ''serotype switching'' may help to overcome this problem. In addition to the Ad serotype 5-based helper virus, Parks et al. have generated a serotype 2 helper virus (72). Helper viruses based on serotypes 1 and 6 have also been generated (Authors' unpublished data). Therefore, genetically identical HDAds of different serotypes can by generated simply by changing the serotype of the helper virus used for vector amplification. Parks et al. demonstrated that mice injected with a serotype 2 HDAd produced serotype 2 neutralizing antibodies, which prevented successful transduc-tion with the same serotype HDAd (72) but did not prevent successful transduction following administration of a serotype 5 HDAd. Successful readministration of HDAd of alternative serotypes has also been demonstrated by Kim et al. (66) (see Section VI.A). As discussed in Section II, there are ~50 human serotypes of Ad. Therefore, it may be possible to create a panel of different serotype helper viruses and use these to generate different serotype but genetically identical HDAds. These HDAds could then be given sequentially every 2 to 3 years when transgene expression wanes from the previous vector administration. Because of the large number of Ad serotypes, this could theoretically continue for the lifetime of the patient, although development of the many helper viruses needed for this approach might prove challenging.

G. Helper Virus Contamination

Currently, the final purity of HDAd preparations is dependent on two enrichment steps. The first is Cre-mediated packaging signal excision during vector amplification. This alone results in about < 1% helper virus contamination (65). If the genome sizes of the HDAd and the helper virus are sufficiently different, further enrichment can be obtained by CsCl ultracentrifu-gation, which can reduce contamination levels to as low as 0.1% or less. Although the final level of helper virus contamination is quite low, further reduction is desirable to minimize any potential toxicity associated with the helper virus, especially in therapies where very high doses of the HDAd may be required. Furthermore, in those cases where the genome sizes of the HDAd and helper virus are unavoidably similar, physical separation of the 2 species by CsCl ultracentrifuga-tion will not be possible and contamination levels of <0.1% will likely be unachievable. Finally, CsCl ultracentrifugation is impractical for large-scale clinical grade vector production. The source of the residual contaminating helper virus that persists during HDAd propagation using the Cre/loxP has been investigated in detail (65). That study revealed that the contaminating helper virus has escaped Cre-mediated packaging signal excision and propagated. Detailed investigations revealed that this was not due to acquisition of Cre-resistant mutations or to the reverse Cre reaction, which would reinsert the excised packaging signal. Nor was it due to inaccessibility of a fraction of the helper viral DNA to Cre. Rather, the results revealed that incomplete packaging signal excision was the result of Cre levels being limiting in the producer cells due to low endogenous levels. Further exacerbating this problem may be Ad-mediated host cell shut off, a well-documented phenomenon in which synthesis of cellular protein is inhibited as a consequence of Ad infection [reviewed in (76)]. The results of this study suggested that further reduction in helper virus contamination may be achieved by increasing the amount of Cre in the producer cells. However, given that very high, constitutive levels of Cre expression have been reported to be cytotoxic in various cell lines, including 293 (77), this strategy may be difficult to achieve.

It is important to ascertain the level of helper virus contamination accurately because high levels of helper virus contamination may affect experimental results and compromise safety. Knowing the level of helper virus contamination also allows for meaningful comparisons between different studies. A variety of methods for determining helper virus contamination have been reported, all of which can be divided into 2 basic categories. The first is based on determining the infectious titer of the contaminating helper virus in an HDAd preparation. These methods included plaque assay on 293 cells (or 293-derived cells) or infectious unit assay, based on the presence of a reporter transgene in the helper virus. Using these infectious titer assays, the level of helper virus contamination is often reported as a percent or a ratio of infectious helper virus in the vector preparation. However, these methods suffer from numerous shortcomings. First, the units of measurement for the HDAd and helper virus are not the same and thus not directly comparable, making it difficult to accurately determine the level of helper virus contamination. Second, it has been shown that infectious assays are 10- to 50-fold less sensitive than DNA-based assays described below, thus grossly underestimating the true level of contamination, and may be incapable of detecting very low levels of helper virus contamination (41,71). Alternative methods of determining helper virus contamination use direct measurement of the amount of HDAd and helper viral DNA in a vector preparation. This has been accomplished by standard Southern blot hybridization analysis (55,71) or quantitative real-time PCR assays (41,56,59). Not only are these methods more sensitive and reliable, but also the unit of measurement is the same for both the HDAd and the helper virus therefore allowing the level of helper virus contamination to be expressed simply as a percentage of the total DNA [% helper contamination = helper DNA/(vector DNA + helper DNA)]. The reader should keep the distinction between the 2 methods of determining helper virus contamination levels in mind when critically reading primary publications. In general, most studies published prior to the year 2000 used infectious titer assays, whereas most (but not all) studies published after the year 2000 used DNA-based methods to determine helper virus contamination levels.

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