Many different promoters have been inserted into Ad vectors and in most cases have retained their activity. Widely used viral promoters include the long terminal repeat of RSV, the SV40 early promoter, and the cytomegalovirus (CMV) major immediate-early promoter. The CMV promoter provides very strong expression of a transgene in many cell types, at least in the short term (Jiang et al., 1996; Guo et al., 1996), and has become perhaps the most widely used promoter in adenoviral vectors. Bartlett et al. (1996) have reported that the constitutively active U1 small nuclear RNA promoter, which is transcribed in essentially all cell types, has activity similar to CMV and is active when placed into an El-deleted Ad vector.
When placed in the adenovirus context, a number of tissue-specific promoters have been shown to be regulated much like their cellular counterparts. Friedman and co-workers (1986) found that the liver-specific rat albumin promoter in an Ad5 vector was expressed in rat or human hepatoma cells or in primary hepatocytes, but not in a myeloma cell line, whereas the B-cell-specific immunoglobulin heavy chain promoter was expressed strongly in myeloma but not hepatoma cells. More recently, studies of a modified mouse albumin promoter driving expression of the human factor VIII gene demonstrated specificity both for hepatocytes in vitro and for liver in vivo (Connelly et al., 1996).
A number of groups have sought to target adenovirus-based gene expression to tumor cells. Hepatoma cells often express elevated levels of a-fetoprotein (AFP) relative to normal liver. A recombinant Ad carrying AFP promoter and enhancer sequences driving the herpes simplex virus thymidine kinase (TK) gene expressed high levels of TK in AFP-expressing tumor cell lines such as HuH-7 or HepG2, but not in non-AFP-expressing liver cell lines such as HLF (Kanai et al., 1996). Other workers have reported that expression of foreign genes placed under the control of the carcinoembry-onic antigen (CEA), osteocalcin (OC), or DF-3 promoters in Ad vectors is specific for CEA-, OC-, or DF-3-expressing tumor cells, respectively (Lan et al., 1996; Ko et al., 1996; Chen et al, 1995). Hashimoto et al. (1996) found that Ad-mediated expression using two different neural cell-specific promoters was restricted to the expected cell types both in culture and after administration of the respective Ad vectors to rat brain in vivo.
The ability to control the level or timing of transgene expression may be desirable in a particular experimental setting. A number of inducible or regulated promoters have been placed into the Ad backbone. For example, a synthetic construct consisting of minimal thymidine kinase promoter sequences and several retinoic acid response elements was inserted into the El region of an Ad5 vector. Transcription of a reporter gene in infected cells could be induced by the addition of retinoids, both in culture and in vivo (Hayashi et al., 1994). The mouse metallothionine promoter also retained its zinc-inducible expression in an Ad context (Yajima et al., 1996). Varley et al. (1995) made Ad constructs containing a luciferase gene driven by either of two inflammation-responsive promoters. Cells infected with either virus were found to express high levels of lu-ciferase in response to the cytokines present in conditioned culture medium. In vivo studies demonstrated that lucif erase expression was upregulated in tissues of mice infected with these vectors and challenged with inflammatory agents such as lipopolysaccharide toxins or turpentine (Varley et al., 1995).
The pattern that emerges from these studies is that cellular promoters in an Ad chromosome often behave as would be predicted from their normal activities, although it may be worth noting that all of these specific promoter activities were generated using insertions into the viral El region. The ability to reproduce cell-specific patterns of gene expression after adenovirus-mediated transfer will be invaluable in targeting transgene function to specific cells and may be useful in optimizing expression of a recombinant protein in the cell type of interest.
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