O I 2 3 4 S 6

Serial Passage Number

Serial passage number B- kb a b Í) 1 2 3 4 5 6 7 8 c

Serial passage number B- kb a b Í) 1 2 3 4 5 6 7 8 c

Figure 3 Amplification of HDAd. (A) Amplification of HDAdC4HSULacZ by serial coinfections of 293Cre cells with the HDAd and the helper virus. HDAdC4HSULacZ contains a LacZ reporter gene (Fig. 2). Therefore, the amount of HDAd produced at each serial passage can be determined by X-gal staining and is expressed as blue-forming units (bfu)/mL. (B) Southern blot hybridization analysis of HDAd amplification from (A). Total DNA was extracted from the coinfected cells at each serial passage, digested with Bgll, and analyzed with a packaging signal probe (see Fig. 2 for details). The amount of HDAd DNA (1.6kb band) increases from passage 0 to a peak by passage 3, consistent with amount of infectious vector produced as shown in (A), whereas the amount of packagable helper viral DNA (1.0-kb band) remains low. Lane a, total DNA extracted from helper virus-infected 293 cells. Lane b, total DNA extracted from helper virus-infected 293Cre cells. Lane c, pC4HSULacZ digested with Bgll and Pmel. (Adapted from Ref. 55.)

Vector genome sizes above the maximum packaging capacity were not efficiently packaged, if at all. Vector genome sizes below 27 kb were inefficiently packaged and frequently underwent DNA rearrangements to produce genomes closer to that of wt viruses (approximately 36 kb) (46,47,62). Therefore, the size of the vector is an important consideration: for efficient packaging and stability, the vector genome should be between ~75% (>27 kb) (47) and 105% (<37.8 kb) (61) of wild-type. Because the minimal Ad c/s-acting sequences and the transgene of interest are usually well below the minimal size required for efficient packaging, the vector must often include ''stuffer'' DNA. The choice of stuffer DNA is important with regard to vector stability, replication efficiency and in vivo performance (56,63,64). Although it remains unclear what constitutes a good stuffer, in general, noncoding eukary-otic DNA has been preferred, whereas repetitive elements and unnecessary homology with the helper virus should be avoided to ensure vector stability and prolonged transgene expression.

In addition to the absolute size of the HDAd, its relative size compared with the helper virus has an impact on the production of (nearly) helper-free vectors. This is because Cre-mediated selection against the helper virus, although efficient, is not absolute (65) (see Section V.G). If the genome size of the HDAd is sufficiently different from that of the helper virus then the 2 species can be physically separated by CsCl ultracentrifugation due to their different buoyant densities. HDAds between 28 and 31 kb have proven ideal because they are efficiently packaged and replicated, as well as easily separated from residual helper viruses (35-37 kb) following CsCl ultracentifugation.

It is also becoming clear that the nature of the transgene has a significant influence on the degree and duration of transgene expression. Specifically, transgenes in their native genomic context have consistently been demonstrated to be superior to their cDNA counterparts with respect to level and duration of expression (64,66,67). This is likely due to a more natural regulation of gene expression. In this regard, because of the large cloning capacity of 37 kb, HDAds offer the advantage of potentially transferring many genes in their genomic context, whereas other vectors (e.g., FG and multiply deleted Ads, retroviral, lentiviral, or adeno-associated viral vectors) cannot due to their limited cloning capacity. In the case of cDNA, expression from a tissue-specific promoter was found to be superior to nonspecific promoters in terms of toxicity and duration of expression (64,68). This has been attributed to a reduction of transgene expression in transduced antigen-presenting cells, following systemic delivery of vectors that carry expression cassettes under the control of tissue-specific promoters (68). Expression from tissue-specific promoters has been demonstrated to be more ''tissue specific'' within the context of an HDAd than a FGAd (69). This may be due to the influence of Ad sequences from the FGAd backbone on transgene expression specificity, which does not occur with HDAds (69). In addition, use of tissue-specific promoters may result in more robust amplification of the HDAd because highlevel transgene expression in the producer cells may negatively impact viral replication during serial coinfections, especially if the transgene product is toxic to the producer cells.

Titration of FG and multiply deleted Ads is straightforward. These vectors can form plaques on monolayers of the appropriate complementing cell lines in a standard plaque assay. The infectious titer can then be determined by enumerating the plaques and expressed as the number of plaque-forming units (pfu)/mL. In addition to infectious titer, the virion particle concentration, based on DNA content, can be readily obtained by measuring the optical density at 260 nm (OD260) of the vector preparation. Comparison of the particle: infectious unit ratio provides a measure of the infectivity of the vector preparation and current Food and Drug Administration standards require a ratio of <30:1 for FG vectors used in

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