L69

permits longer transgene expression (51,91,92). However, the temperature-sensitive mutation is unstable and partially replication competent at 37°C.

Other mutations in early genes have been used in Ad vectors, including partial and complete E2 and E4 deletions. Both of these genes are essential for viral replication and therefore necessitate the production of cell lines that complement both the E2 or E4 deletion, as well as the E1 deletion. In general, this has been achieved using 293 cells transfected with the appropriate E2 or E4 gene driven by an inducible promoter (Table 1). In a typical example, E4 deleted vectors were constructed in a cell line that expressed the E4 OFR6 behind an inducible metallothionein promoter (93). Cell lines of this type are more difficult to work with than 293 cells, and the efficiency of vector production is often lower. There is inconsistent data on whether additional genomic deletions result in a blunted immune response and whether this translates into longer persistence of transgene expression. Some studies are complicated by the immune response to the foreign transgene and to the vector, and by the tendency of the commonly used CMV promoter to be inactivated over time without vector elimination. For example, one report (94) indicated that a complete E4 deletion has no effect on the time course of gene expression in immunocompetent animals after administration to lung or liver. This is at variance with other reports (50,95,96) showing that E4 deletion results in a reduced immune response and longer transgene expression. The details of vector construction, route of administration, promoter used (97), dose, and genotype of the recipient are critical to the efficacy of E4 (and other) deletion(s). It is likely that studies in humans will be necessary to determine if additional genomic deletions have an impact of the duration of expression of the therapeutic gene and whether this translates into a significant clinical impact.

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