Novel strategy for the generation of targeted toxins for glioma therapy using regulated expression and Cre recombinase

PUNTEL; MONDKAR; XIONG; KROEGER; LOWENSTEIN; CASTRO

Los Angeles, California

Congreso; American Association for Cancer Research; 2007

Novel strategy for the generation of targeted toxins for glioma therapy using regulated expression and Cre recombinasePuntel M., Mondkar S., Candolfi M., Xiong W., Kroeger K.M., Lowenstein P. R., and Castro M. G.Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, and Department of Medicine, and Department 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 incurable brain tumor for which novel therapies are needed. A promising approach is to use toxins fused to cytokines which will bind and be internalized only into GBM cells. This constitutes a targeted cytotoxic approach. One such approach already in clinical trails is IL13 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 side effects if delivered systemically. We hypothesized that IL13-PE could be encoded within regulatable adenoviral vectors with the goal of minimizing the toxic effects of the toxin to the producer cells. Our approach consists of a Doxicycline-regulatable gutless adenovirus vector carrying a LoxP flanked STOP sequence at the N-terminus of IL13-PE, abolishing its expression. The expression of the mIL13-PE fusion protein is achieved only 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 (IL13-PE) downstream of the STOP sequence, generating pBS302.IL13-PE (5.4kb). We then subcloned the STOP.IL13-PE cassette into an intermediate vector bearing mutated IL4, used as a safety feature to block any putative binding of IL13-PE to the IL4/IL13 receptor present in normal surrounding brain cells. The resulting fragment encoding IL4-TRE- STOP-IL13-PE was cloned into the plasmid encoding the TetOn switch, generating the pBMCS1.Kana-TetON-IL4.TRE.STOP.IL13-PE (14.6kb). This entire cassette was then cloned into the gutless adenoviral plasmid, i.e., (pSTK.TetON.IL4.TRE.STOP.IL13-PE) (34.4kb). Our results show regulated expression of IL4, provided by the TetOn switch; activation of IL13-PE expression provided by the removal STOP sequence by Cre recombinase; and GBM-specific toxicity of IL13-PE.This provides a safe and powerful strategy to treat GBM based on the following premises: 1- Use of a targeted toxin (IL13-PE) that will limit undesired toxicity to the surrounding normal brain, 2- Delivery using a gutless adenoviral vector that will avoid the elimination of therapeutic gene expression due to the presence of adenovirus-specific immune response that is very frequent in the human population, 3- Regulatable expression of IL4 and IL13-PE allows switching expression ON and OFF depending on clinical need, and 4- the presence of a STOP sequence flanked by LoxP sites that completely abolishes the expression of the toxin in the absence of the Cre recombinase. The above features the safety and specificity of this approach which could be translated into human trials.Funded by NINDS 1RO1 NS 42893.01, U54 NS045309-01, 1R21 NS047298-01; and Bram & Elaine Goldsmith Chair in Gene Therapeutics to P.R.L.; NINDS 1R01 NS44556.01, and NIDDK 1 RO3 TW006273-01 to MGC; The Linda Tallen & David Paul Kane Foundation and the Board of Governors at CSMS.