927. Development of Non-Viral Gene Therapy Targeting Insulin-Like Growth Factor (IGF) System for Treatment of Brain Tumors
Authors: Leah R. Villegas, Sean Sullivan, Maria Grant
Journal: Molecular Therapy (2006)

This project applied IGF1 receptor (IGF1R) silencing and IGF binding protein (IGFBP3) expression to target the IGF1 signaling pathway in brain tumors. The objectives of this project were to: (1) characterize the role of the IGF system in rat glioma (RG2) cells by utilizing a drug-inducible stable cell line that expresses an anti-IGF1 receptor ribozyme (IGF1R-Rz) (2) investigate a therapeutic approach of the IGF1R-Rz by cationic lipid mediated transfections (3) characterize the impact of IGFBP3 expression on brain tumor cells. A stably transformed RG2 cell line (Fisher rat glioma) was developed to express an anti-IGF-1 receptor ribozyme (IGF-1RRz) under the mifepristone-inducible Gene Switch (Invitrogen). A β-galactosidase reporter gene was first used to validate Gene Switch expression where mifepristone-induced cells showed 20-fold greater expression compared to non-induced cells. Expression of the IGF1R- Rz from this inducible expression system showed complete arrest of cell proliferation, while non-induced cells continued to proliferate. Western blots confirmed that mifepristone induction of ribozyme expression decreased the amount of IGF-1 receptor by 89% compared to non-induced cells. To investigate a more therapeutic approach, RG2 cells were transfected with IGF1R-Rz in a chick beta actin promoter expression plasmid using cationic lipid mediated delivery. In a population where approximately 20% of cells uptake the IGF1R-Rz expression plasmid, a 20% inhibition of cell proliferation was attained 72 hours after transfection. Expression of secreted IGFBP3 to bind IGF1 was selected for the following reasons: (1) IGFBP3 has a higher binding affinity for IGF1 than for its receptor, (2) a secreted protein impacts surrounding cells so inhibition of cell proliferation will not be restricted only to the transfected cells and (3) IGFBP3 induces an IGF-independent apoptotic pathway. The coding sequence for rat IGFBP3 was obtained by PCR from rat liver cDNA and cloned into a CMV promoter expression plasmid. Cationic lipid mediated delivery of the IGFBP3 plasmid was used for transient transfections into RG2 cells which demonstrated 20% inhibition of cell proliferation 72 hours after transfection. The present studies have identified the IGF1 system as a potential target for treating brain tumors. Elimination of the IGF1R in a stable cell line illustrated the significance of IGF1 signaling in glioma cell growth and expression of IGF1R-Rz and IGFBP3 through cationic lipid mediated delivery show therapeutic value. Concurrent studies include further characterization of IGF1 downstream signaling such as IGF1R/AKT phosphorylation and characterization of IGFBP3- induced apoptosis. Future studies will include development of protease-resistant IGFBP3 to increase efficacy because the tumor microenvironment contains proteases making IGFBP3 susceptible to proteolysis, allowing free IGF1 ligand to interact with its receptor. Once characterized, co-expression of IGF1R-Rz and IGFBP3 will be used to demonstrate possible cumulative effects that utilize two different targets of the IGF system.