Another major adenoviral targeting strategy is to control the expression of the therapeutic gene by using transcriptional regulatory elements (see Fig. 3). In the human genome, genes are controlled by distinct promoters (45), which are applicable for the control of transgene expression from an adenovirus vector (46).
The most straightforward way is to place the specific promoter in the 5'-upstream region of the transgene placed in the vector. The transcription cassette with the specific promoter is usually placed in the El-deleted region in both directions (47). Various transgenes can be incorporated, including reporter genes (e.g., luciferase, P-galactosidase, fluorescent proteins), prodrug activating enzyme (e.g., thymidine kinase, cytosine deaminase). If the adenovirus El region (required for replication) is placed under the control of any exogenous promoter, the vector can achieve conditional replication depending on the promoter activity (18,48,49). However, many factors (e.g., intrinsic transcription activation activity and enhancer elements of vector backbone, removal of distant control elements during cloning, and absence of histones) may possibly affect promoter profiles after incorporation in an adenoviral vector system. The most important consideration is that not all promoters showing a suitable profile in plasmid-based experiments will maintain the desired selectivity and strength in an adenovirus vector configuration.
A second way of transcriptional targeting is to use artificially modified or structured promoters to achieve the desired control. In general, promoters consists of core promoter elements including transcription initiation sites, and number of enhancer elements in up- or down-stream of the core region. Those enhancer regions may exist far upstream or sometime in the intron of the gene itself. To configure these elements (50,51) or to create new promoters with the required transcription selectivity (52), elements from one or multiple promoters may have to be combined into artificial promoters. This method has big potential for the development of custom-designed transcription profiles. On the other hand, because the activity of newly created promoters is not completely predictable, it has the potential risk to lead to aberrant expression (e.g., unexpected high activity in normal organs, leakiness resulting from the removal of unidentified negative control elements, and so forth). The establishment of appropriate promoter design strategies and high efficient methods for screening are necessary to advance the development of this strategy for Ad targeting.
An extension of using promoters for Ad transcriptional targeting is the application of inducible promoters for the regulation of the expression. There are several promoters known to be inducible by an external nontoxic stimuli (e.g., tetracycline ) or radiation (54). These promoters are useful should post-administrative regulation of transgene expression be necessary. Also, some vectors which encode highly toxic genes may need negative regulation of expression during viral amplification in order to avoid effects on virus replicaiton (55,56).
Another approach in transcriptional targeting is based on a binary system. This system uses a specific promoter to express a triggering protein which binds to a regulatory element that turns on the transcription of an extrinsic gene (57). Similar effect is also achievable by using the bactriophage RNA polymerase and its recognition sequence (58) or with CRE recombinase and its recognition sequence (lox-p) (59). This strategy is extremely useful for constructing an expression system to enhance "selective but weak" promoters although the vector structure and regulation mechanism are more complex than the use of conventional strategies (60).
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