AdMax™ – Hi IQ for High Protein Expression From Adenoviral Vectors
It is possible to enhance expression of proteins cloned in Ad vectors by introducing an intron between the transcription start and the translation start of the protein. However, high levels of transgene expression can then have inhibitory effects on virus replication, even with proteins that are not cytotoxic when expressed at moderate levels. Further, mild inhibition of virus replication can significantly reduce the efficiency or prevent rescue of vectors, given that rescue from transfected or cotransfected plasmid DNA is inefficient. Consequently, isolation of high expression vectors designed for certain proteins fails. It is difficult to predict which proteins can be driven to high expression, but the problem is common in the expression of glycoproteins and DNA binding proteins or enzymes.
The inhibition of protein expression, even if only partial, can significantly improve the rescue efficiency of cassettes containing a cDNA expressed from a promoter-intron combination (DA Matthews et al.; Cummings and Graham, unpublished). In cell lines that express the lac repressor, and expression cassettes that contain the lac operator (repressor binding sequence), transcription from the cassette is inhibited when the vector is rescued or propagated in host cells that express the repressor. Unlike other gene regulation systems, high level expression in normal cells or in vivo does not require a second transactivator.
This “Hi-IQ” system has been combined with the AdMax™ two plasmid vector rescue system for isolation of vectors expressing all but the most toxic proteins at very high levels (see Figure). The system differs from the original AdMax™ in its use of 293 cells expressing lac repressor (293-IQ cells) and shuttle plasmids containing expression cassettes with the MCMV IE gene promoter, an intron, and a lac operator sequence for regulation of expression. The vectors are rescued and propagated in 293-IQ cells and the transgene can then be expressed by transduction of any other cell type where transcription is relieved of repression.