FOREIGN GENE-EXPRESSION BY HUMAN ADENOVIRUS TYPE 5-BASED VECTORS STUDIED USING FIREFLY LUCIFERASE AND BACTERIAL BETA-GALACTOSIDASE GENES ASREPORTERS

Adenovirus (Ad) vectors have been used extensively to obtain high-leve l expression of foreign genes in mammalian cells and are currently bei ng studied for use as live viral-vectored vaccines and as gene transfe r vectors for gene therapy. Many Ad recombinants have been generated t hat express foreign genes inserted in early region 3 (E3); however, li ttle has been done to study the importance for gene expression of regu latory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian v irus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.8 8 or 2.69 kb. The greatest levels of luciferase expression were obtain ed with a vector containing the luciferase gene under the control of t he SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 d eletion. In contrast, LacZ expression was highest with a vector contai ning the LacZ gene with just the SV40 polyadenylation sequence combine d with a 1.88-kb 53 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DN A replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-spe cific mRNA for various vectors in each series did not vary significant ly. These results indicate that the kinetics and efficiency of express ion of genes inserted into the E3 region, in nonconditional helper-ind ependent vectors, may be more strongly dependent on the sequences in t he foreign gene insert itself than on flanking regulatory sequences su ch as those used here, derived from SV40. (C) 1995 Academic Press, Inc .