Gliobastoma is a devastating brain tumor, for which there is no cure; patients succumb between 6 to 12 months after their diagnosis. Human glioma cell lines and more than 80% of gliomas in situ express high amounts of an IL-13 receptor (IL-13a2R), different from that expressed in normal tissues (IL13R/IL4R), a feature that can be used to target molecules to glioma cells without affecting the surrounding brain tissue. To this end, human IL-13 (hIL-13) has been fused to a mutated form of Pseudomonas exotoxin (PE). After receptor-mediated internalization into IL13a2R-expressing glioma cells, it inhibits protein synthesis, leading to cell death. A mutant of hIL-13, i.e., IL13.E13K (muIL-13), has shown negligible binding to normal brain cells and higher affinity for the glioma-specific IL-13 receptor than wild type hIL-13. Our goal is to construct a high capacity adenoviral vector (HC-Adv) encoding the chimeric toxin, muIL-13-PE. To increase the safety, the vector will also encode the cDNA of a mutated IL-4, i.e., IL4.Y124D (muIL4), an antagonist of the IL13R/IL4R present in normal cells; which does not interact with IL13a2R expressed in glioma cells, nor does it affect the cytotoxicity of the chimeric toxin. Further, we will incorporate the regulatory TetON system into the viral vector which will allow tight regulation of expression of the therapeutic transgene, by switching it """"""""on"""""""" and """"""""off"""""""" by the addition or withdrawal of doxycicline (DOX). The effects of muIL-13-PE expressed by transduced cells will be assessed in vitro in human glioma cell lines and in vivo in the normal brain. We will perform dose-escalation studies following administration of the HC-Adv vectors encoding the targeted toxin within the brain of immune competent mice. We will also asses its therapeutic efficacy in intracranial human gliomas implanted in nude mice. We will determine expression levels, therapeutic efficiency, survival, side effects, and persistence of the viral vector genomes within the tumor and adjacent brain tissue in vivo. Considering the putative side effects reported after the systemic administration of the chimeric toxin, we hypothesize that the administration of the HC-Adv vector expressing muIL-13-PE, to glioma bearing mice will lead to effective intratumoral levels of the high affinity chimeric toxin, killing glioma cells but preserving normal brain tissue. Previous data provide strong evidence that HC-Adv vectors will exert long term, regulatable expression of the toxin even in the presence of systemic anti-adenoviral immunity as could be present in humans. Expression muIL4 will further prevent muIL13-PE binding to normal surrounding brain cells without affecting its cytotoxicity on glioma cells, adding a significant safety feature to our approach. Finally, the introduction of the TetON regulatory system will allow tight regulation of expression of the encoded chimeric toxin. In summary, we expect to develop a targeted glioma gene therapy approach with high therapeutic efficacy and negligible toxicity which could be translated into clinical trials. ? ? ?
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