Drugregulated Promoters

For many applications, it may be desirable to regulate expression of a therapeutic transgene in vivo, either to limit toxic effects from high-level expression or more closely resemble physiological expression profiles. We created a viral vector with regulatable transgene expression by using an autoregula-tory loop that consisted of a promoter with 5 tandem copies of the 17-bp Gal4 DNA recognition element that could be trans-activated by vector-encoded chimeric Gal4/VP16 protein (Fig. 7A), based on the ability of the chimeric protein to transactivate promoters containing this site (152-154) despite the repressive presence of nucleosomes (155,156). The constitutive Gal4/ VP16 transactivator was able to induce transgene expression from a Gal4-sensitive minimal promoter in the background of the virus (115). Regulation was achieved by replacing the constitutive transactivator with a chimeric molecule consisting of the hormone-binding domain of the mutated progesterone receptor fused to the transactivation domain of VP16 and DNA-binding domain of Gal4 (157,158). In the presence of the progesterone analog RU486, the inactive chimeric transactivator assumes a conformation allowing it to bind to and transactivate the Gal4 recognition site-containing promoter driving transgene expression. Compared with control (Fig. 7B), the completion of the autoregulatory loop following administration of RU486 (Fig. 7C) resulted in substantial enhancement of expression of the transgene in the CNS (115) (Fig. 7D). Following infection of the rat hippocampus with the regulatable virus, levels of viral vector-derived transgene expression are stimulated by administration of the inducing agent RU486 (159).

Figure 7 In vivo regulated transgene activation from HSV vectors by drug-inducible recombinant transactivator (RTA). (A) The GLVP RTA vector contains the inducible chimeric transactivator GAL4:VP16:HBD, composed of the yeast GAL4 DNA-binding domain fused to the HSV VP16 transactivation domain fused in frame to a mutant form of the progesterone receptor hormone-binding domain, which can be activated by RU486. In addition, the vector contains a promoter-reporter cassette (GAL45TATA-lacZ) that is responsive to activation by the transactivator (RTA). (B) In the absence of the drug, the GAL4:VP16:HBD recombinant transactivator cannot bind to the GAL4 sites in the minimal TATA box promoter, resulting in no expression of the p-galactosidase transgene. (C) Following administration of RU486, the tripartite transactivator undergoes a conformational change that allows the RTA to bind to the GAL4 binding sites to yield p-galactosidase expression. (D) Quantitation of p-galactosidase transgene expression in the presence of RU486 displays activation of the transgene promoter by the inducible transactivator following injection of the vector into rat CNS.

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