The first-generation adenoviral vectors discussed so far are not suitable to obtain long-term gene expression. This is generally attributed to downregulation of the CMV promoter and to an immune response to virus-infected cells. These infected cells are recognized by the immune system since some of the adenoviral backbone genes, despite the absence of the early regulator switch E1, are clearly expressed. If the expressed transgene is also highly immunogenic (such as the bacterial lacZ gene), this will further contribute to the immune response.
To prolong gene expression after adenovirus-mediated gene transfer, systemic immune suppression has been applied (15,16). In addition, adenoviral vectors have been modified to prevent late adenoviral gene expression. These viral vector modifications consist of progressive deletions of the adenovirus backbone and thus more effective crippling of the virus.
Second-generation adenoviral vectors are based on the observation that the adenovirus mutant TS125 only grows at 32°C owing to a temperature-sensitive mutation in the E2 protein. The vector system designed from this E2 mutant thus consists of an adenovirus backbone plasmid containing the TS-E2 variant. The adenoviral vectors generated from this plasmid can be amplified at 32°C but not at 37°C. Thus, in the liver at the mouse body temperature of 37°C, these vectors will be double deficient (AE1A and TS-E2). Using these vectors, prolonged expression times have been reported (17,18).
Helper-dependent or gutted adenoviral vectors lack all endogenous adenoviral genes (19). The most successful system is based on a helper adenoviral vector that is E1A-deficient and in which the packaging signal is surrounded by LoxP sites (floxed). This helper vector can be amplified in regular 293 or 911 cells. The gene of interest is cloned in a vector flanked on both sides by inverted terminal repeats and on one side by a functional packaging signal. Inserts of up to 36 kb can be cloned in this "gutted" vector.
The helper-dependent vector is amplified on a 293 cell line that expresses the Cre-recombinase protein. Upon infection of this cell line with the floxed helper vector, the packaging signal is deleted and the helper DNA will not be packaged into the virion. Transfection of the linearized vector with a functional packaging signal will result in preferential packaging of this vector into the virion. Long-term expression has been demonstrated with helper-dependent vectors (20).
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