Adenoviral mediated gene transfer of Pseudomonas exotoxin fused to IL-13 to treat experimental glioblastomas

CANDOLFI; XIONG; MUHAMMAD; PUNTEL; LIU; KROEGER; MONDKAR; LERNER; LEE; FOULAD; DEBINSKI; RODRIGUEZ; LOWENSTEIN; CASTRO

Boston, Massachusetts, USA

Congreso; American Society of Gene Therapy; 2008

Abstract #3324Adenoviral mediated gene transfer of Pseudomonas exotoxin fused to IL-13 to treat experimental glioblastomasMarianela Candolfi, Weidong Xiong, Mariana Puntel, Chunyan Liu, Kurt Kroeger, Sonali Mondkar, Ronald Rodriguez, Pedro Lowenstein and Maria CastroCedars Sinai Medical Center, Los Angeles, CA, Johns Hopkins University School of Medicine, Baltimore, MDSince Glioblastomas (GBM) overexpress IL132R, an IL-13 receptor that is absent in the normal brain, attempts have been made to target toxic compounds to glioma cells by fusing them with IL-13. Considering that protein formulations have a short half life and could require frequent administrations to be effective, we aimed to use adenoviral vectors (Ads) to express the chimeric toxin composed by IL-13 and Pseudomonas exotoxin A (PE) in human glioma cells in vitro and in vivo. We constructed an adenovirus, Ad-IL4-TRE-IL13.PE expressesing IL-13-PE, and, as a safety feature, a mutated IL-4 that blocks any putative binding of the toxin to the physiological IL13/IL4R. Transgenes’ expression is driven by the bidirectional TRE promoter, which is activated by the transactivator (TetON, expressed within Ad-TetON), in the presence of the inducer, i.e., Dox. Considering that during viral replication transgenes are expressed in vector-producing cells, we developed Ads encoding IL-13-PE toxin in DTR-293 cells that are resistant to the toxin. DTR-293 cells are stably transfected with pHED-7, which encodes the gene for ADP ribozilation-resistant elongating factor-2 (EF-2) from CHO cells. IL-4, IL-13 and PE expression were detected human U251 glioma cells infected with Ad-IL4-TRE-IL13-PE+Ad-TetON, which reduced cell viability 70% when the human glioma cells were incubated in the presence of in the "ON" state (Dox+). Human glioma cell viability remained unaffected in the "OFF" state, indicating that the expression of the chimeric toxin can be tightly regulated. Transgene expression from Ad-IL4-TRE-IL13-PE was also detected in COS-7 cells. However, COS-7 cells, which do not express the IL13alpha2R, did not undergo cell death in the presence of the therapeutic virus, suggesting that IL-13-PE cytotoxicity is specific to glioma cells. We also administered Ad-IL4-TRE-IL13-PE+Ad-TetON in the striatum of nude mice, which were fed chow containing Dox. IL-4, IL-13 and PE toxin were readily detected in the mouse brain, with no signs of toxicity. Then, we administered Ads intratumorally into intracranial human U251 GBM xenografts in nude mice. Ad-IL4-TRE-IL13-PE significantly increased the survival of tumor-bearing mice when compared to saline-treated (45 days) or mice treated with the control virus (Ad-IL4-TRE-IL13, 53 days). Our results suggest that Ad-mediated intratumoral expression of IL13-PE toxin will lead to effective cytotoxicity in IL-132R expressing-GBM cells without side effects to the surrounding non-neoplastic brain.Supported by: NIH/NINDS 1R01 NS44556.01, Minority Supplement NS445561; NIH/NINDS 1 RO3 TW006273-01 to M.G.C.; NIH/NINDS 1 RO1 NS 42893.01; U54 NS045309-01, 1R21 NS047298-01, the Bram and Elaine Goldsmith and the Medallions Group Endowed Chairs in Gene Therapeutics to PRL and MGC, respectively, and The Linda Tallen & David Paul Kane Annual Fellowship and Board of Governors at CSMC