Novel Strategy for the Generation of Targeted Toxin Pseudomonas Exotoxin A Fused to Mutated (mu) hIL-13 for Glioma Therapy Using Regulated Expression and Cre Recombinase.

XIONG; PUNTEL; CANDOLFI; LIU; KROEGER; HERSHMAN; KHAYZNIKOV; LOWENSTEIN; CASTRO

Boston, Massachusetts, USA

Congreso; American Society of Gene Therapy; 2008

Novel Strategy for the Generation of Targeted Toxin Pseudomonas Exotoxin A Fused to Mutated (mu) hIL-13 for Glioma Therapy Using Regulated Expression and Cre Recombinase.Weidong Xiong1,2, Mariana Puntel1,2,Marianela Candolfi1,2, Chunyan Liu1,2, Kurt Kroeger1,2, Harvey Herschman2, Maksim Khayznikov1,2, Pedro R. Lowenstein1,2, Maria G. Castro1,21Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, and Department of Medicine, and 2Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, 8700 Beverly Blvd, Davis Bldg, Rm 5090, Los Angeles, CA 90048. castromg@cshs.orgGlioblastoma (GBM) is an aggressive brain tumor for which novel therapies are needed. A promising anti-GBM therapeutic approach is to use toxins fused to cytokines which will bind to GBM specific receptors and thus will be internalized only into GBM cells. One such approach already in clinical trails is wild type hIL13 fused to domains II and III of Pseudomonas aeruginosa exotoxin A (PE). One shortcoming of the protein formulation is its short half life and its putative adverse side effects. We hypothesized that muIL13-PE could be encoded within regulatable adenoviral vectors with the goal of minimizing the toxic effects of the toxin to the producer cells. We developed a doxycycline-regulatable adenovirus vector carrying a LoxP flanked STOP sequence at the N-terminus of the muIL13-PE construct, abolishing its expression. The expression of the muIL13-PE fusion protein is achieved after the excision of the STOP sequence by co-expression of Cre recombinase. To do this, we cloned a fusion protein consisting of mutated IL13 and domains II and III of PE toxin (muIL13-PE) downstream of the STOP sequence, generating pBS.MCS1-STOP.muIL13-PE. We then subcloned the STOP.muIL13-PE cassette into pdeltaE1sp1A vector to generate shuttle vector pdeltaE1sp1A [IL4-TRE- STOP.muIL13-PE], which also encodes mutated IL4, used as a safety feature to block any putative binding of muIL13-PE to the IL4/IL13 receptor present in normal surrounding brain cells. 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. Using ELISA and immunocytochemistry, muIL-4, muIL-13 and PE expression were detected in COS7 cells and human U251 glioma cells infected with Ad-muIL4-TRE-STOP.muIL13-PE+Ad-TetON and Ad-CAG-Cre, when the cells were incubated in the presence of Dox. muIL13-PE is expressed only in the presence of the inducer DOX and Cre which excises the STOP sequences. Our results show regulated expression of muIL4, provided by the TetOn switch; and of muIL13-PE provided the TetON switch in combination with the removal STOP sequence elicited by Cre recombinase. Our results also demonstrate GBM-specific toxicity of muIL13-PE. This provides a safe and powerful strategy to treat GBM based on the following premises: 1- Use of a targeted toxin (muIL13-PE) that will limit undesired toxicity to the surrounding normal brain, 2- Regulatable expression of muIL4 and muIL13-PE allows switching expression ON and OFF depending on clinical need, and 3- the presence of a STOP sequence flanked by LoxP sites that completely abolishes the expression of the targeted toxin in the absence of the Cre recombinase. Funded by NIH-NINDS grants and The Linda Tallen & David Paul Kane Foundation.3 Key words not included in the abstract title: Cancer gene therapy; Gene expression; Adenovirus