Cytoplasmic expression of EGFR shRNA using modified T7 autogene-based hybrid mRNA/DNA system induces long-term EGFR silencing and prolongs antitumor effects

Abstract

Unusual activation or overexpression of epidermal growth factor receptor (EGFR) has been found in various cancers, and therefore down-regulation of EGFR expression is recognized as a promising strategy for cancer treatment. For decades, RNAi has emerged as an effective solution to regulate gene overexpression, but transient effects of exogenous siRNA have limited the development of EGFRtargeting siRNA therapeutics. Recently, we developed T7 autogene-based hybrid mRNA/DNA system as a non-viral vector for shRNA production and reported the feasibility of long-term silencing for RFP expression as a concept of proof. To investigate its therapeutic availability in cancer therapy, we here modified the hybrid system to stably express EGFR shRNA and confirmed the antitumor effects. When autocatalytic production of T7pol protein in cytoplasm was combined with pT7-driven cytoplasmic transcription for EGFR shRNA, a single transfection lead to stable EGFR silencing in SKOV3 cells for more than 13 days. Also, liposomal systemic administration at two-week intervals resulted in significant inhibition of tumor growth in both SKOV3-bearing mice and PDX models, contrast to the conventional siRNA approach. Our results show an efficient strategy to overcome the temporary effects of synthetic EGFR siRNA in cancer treatment and ultimately provide a potential candidate as an anticancer drug.