Gliobastoma is the most devastating brain tumor in adults and none of the conventional anticancer therapies has been successful in significantly prolonging the lifespan of the patients. More than 80% of gliomas in situ express high amounts of a mutated IL-13 receptor (IL13a2R), different from the one expressed in normal cells. A chimeric toxin consisting of IL-13 fused to a mutated Pseudomonas exotoxin, (PE) has been shown to kill IL-13a2R expressing cells. PE lacks its endogenous protein binding domain, which allows it to bind to the ubiquitous expressed ct2-macroglobulin receptor, but retains its ability to inhibit protein synthesis, which leads to cell death. Thus, its fusion to IL-13 retargets the cytotoxic molecule to IL13a2R-expressing glioma cells. Mutants of IL-13, such as IL13.E13K (mulL-13), have shown negligible binding to normal cells and higher affinity to the glioma-specific IL13a2R when compared to IL-13. However, the systemic treatment with the chimeric toxin requires high concentrations that can induce adverse side effects while the intratumoral treatment requires repeated administration due to the short half life of the toxin. In spite of these limitations this approach is currently considered one of the most exciting novel treatments for this devastating disease, and has provided very encouraging results in early clinical trials performed at various clinical centers. We hypothesize that intratumoral administration of a high-capacity adenoviral vector (HC- Ad) expressing the chimeric protein mulL-13 fused to PE, to human glioma bearing nude mice, will lead to effective intratumoral concentration of the chimeric toxin, killing human glioma cells but preserving normal brain tissue. HC-Ad vectors have the advantage of high cloning capacity and, due to their minimal antigenicity, they elicit stable transgene expression in vivo with low toxicity. To increase the specificity of our approach, we will introduce into the vector the cDNA of mutated IL-4, (mulL4). mulL4 acts as an antagonist, binding to IL13R/IL4R present in normal cells, but it does not interact with IL13a2R expressed on glioma cells, nor does it affect the cytotoxic effects of the chimeric toxin in GBM cells. To further improve the safety of this treatment we incorporated the regulatory TetON system into the vector that allows tight regulation of expression of the therapeutic transgene, by switching it """"""""on"""""""" and """"""""off"""""""" as and when needed by the addition or withdrawal of doxycycline (DOX). Our previous data provide strong evidence that HC-Ad vectors provide long term, regulated expression of the chimeric toxin even in the presence of systemic anti-adenoviral immunity as present in humans. Thus, we hypothesize that the targeted glioma gene therapy approach proposed will elicit high therapeutic efficacy in the absence of adverse side effects. ? ? ? ?
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