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In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
Published on: May 5, 2023
Cytoplasmic expression of EGFR shRNA using a modified T7 autogene-based hybrid mRNA/DNA system induces long-term EGFR
Sharmin Seraj1, Young Jae Cho2, Jeong-Won Lee2
1Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul 136-791, Republic of Korea; Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 136-791, Republic of Korea.
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 EGFR-targeting 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.
Insights
A novel hybrid mRNA/DNA system stably silences epidermal growth factor receptor (EGFR) for over 13 days, offering a promising cancer therapy. This approach overcomes limitations of traditional siRNA, showing significant tumor growth inhibition in preclinical models.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Epidermal growth factor receptor (EGFR) overexpression drives various cancers, making its downregulation a key therapeutic strategy.
- Conventional RNA interference (RNAi) using small interfering RNA (siRNA) faces limitations due to transient gene silencing effects.
Purpose of the Study:
- To develop and evaluate a novel T7 autogene-based hybrid mRNA/DNA system for stable, long-term silencing of EGFR in cancer therapy.
- To assess the therapeutic efficacy of this system in preclinical cancer models.
Main Methods:
- A modified T7 autogene-based hybrid mRNA/DNA system was engineered for stable expression of EGFR shRNA.
- Cytoplasmic transcription of EGFR shRNA was driven by pT7, coupled with autocatalytic T7 RNA polymerase production.
- Systemic administration via liposomes was performed in SKOV3-bearing mice and patient-derived xenograft (PDX) models.
Main Results:
- Single transfection achieved stable EGFR silencing in SKOV3 cells for over 13 days.
- Liposomal administration at two-week intervals significantly inhibited tumor growth in both mouse models.
- The hybrid system demonstrated superior long-term silencing compared to conventional siRNA approaches.
Conclusions:
- The T7 autogene-based hybrid mRNA/DNA system provides an effective strategy for sustained EGFR silencing.
- This approach overcomes the transient nature of synthetic siRNA, presenting a potential therapeutic candidate for EGFR-driven cancers.
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