Use of Adenoviral Vectors with a Minimal Cytomegalovirus Promoter

E1-deleted recombinant adenoviruses 5 are widely used as gene vectors for basic and clinical research. Most adenoviral constructions bear an expression cassette inserted into the E1 region of their genome close to the 5′ end and include a heterologous promoter. The major immediate early human cytomegalovirus (hCMV) and the Rous sarcoma virus promoters, which have fairly high and consistent activity, are among the most extensively used. Nevertheless, expression of delivered protein from these promoters is often excessive, and weaker transcriptional activity would be desirable. Adenoviral sequences located 5′ of the site of insertion, in the region between nucleotides 22 and 341, contain a number of identified enhancer elements (the left ITR and the E1A enhancer) that, in certain conditions, may have an intrinsic promoter activity or may modulate the activity of inserted heterologous promoters (1–4). At 3′ of the insertion site there are enhancer sequences such as that of the right ITR and those present in the E2 and E4 promoters, which, although quite distal, can also interfere. This concept is supported by the inclusion of insulator sequences from the chicken γ-globin locus flanking the expression cassette (4), which abolished the background expression of a metalinducible promoter and increased the response to inducible factors. Therefore, characterization of the influence of adenoviral sequences on weak heterologous promoters is of high interest for the design of new adenoviral vectors. The objective of this study was to reveal the activity of these viral sequences, in the context of hepatic and muscle cells, using the minimal region of the major immediate early hCMV promoter. Two adenoviruses were prepared: AdMIN and AdCMV. AdMIN included a minimal hCMV promoter also used by Baron et al. (5) (-31 to +16 nucleotides from the start transcription site) cloned at nucleotide 455 of adenoviral genome in the backbone of plasmid pACCMVpLpA (6), followed by the reporter gene EGFP. In AdCMV, which was prepared for comparison purposes, the immediate early hCMV promoter (-806 to +16 nucleotides from the start transcription site) was cloned instead of MIN. Adenoviruses were generated by standard techniques (6) and used to transduce several immortalized hepatic cells including FTO2B, H4IIE, Hep3B, HepG2, and primary cultured rat hepatocytes. Gene delivery to cell cultures was accomplished by exposing cells to viruses for 2 h at a MOI of 80 to ensure high efficiency of transduction. Infections were always performed in parallel with both viruses on each cell type. After two days of culture, cells were scraped in Tris buffer, sonicated, and centrifuged at 7400× g for 15 min. Protein aliquots (1.5 and 15 μg) of the supernatants were run on a SDS-polyacrylamide gel and transferred into hydrophobic filters. Ponceu S staining was routinely used to confirm proper protein loading and uniform sample transfer to the membrane. EGFP immunodetection was performed by regular Western blot procedures. In AdCMV-transduced cells transcriptional activity of the hCMV promoter proved to be high, although slight differences were observed among cell types. In the H4EII cell line and primary cultured hepatocytes, protein levels were about half of those in the rest of the cells (Figure 1). When AdMIN was used to express EGFP, much higher variability was detected. EGFP levels were negligible in rodent hepatoma cells, FTO2B and H4IIE, but significant in human hepatic cell lines and rat hepatocytes. In these cells, however, transcriptional activity from AdMIN was about 10 times lower than that from AdCMV (Figure 1). Expression of EGFP from AdMIN and AdCMV was also tested in primary cultured human muscle cells (obtained with informed consent, Hospital Vall d’Hebron, Barcelona) and in the C2C12 immortalized rodent cell line. Myoblasts and myotubes were analyzed four days after adenoviral transduction. In all these cells, both myoblasts and differentiating myotubes, EGFP expression was high when using the AdCMV construction (Figure 2). In contrast, EGFP protein levels were barely detectable in C2C12 cells and absent in primary cultured muscle cells transduced with AdMIN. The differences in AdCMV activity Benchmark