Targeted silencing of master transcription factor SPDEF to reduce mucus production in airway diseases by epigenetic editing

Background: Airway mucus hyperproduction contributes to the morbidity and mortality in asthma and COPD. Reducing mucus production is crucial for improving patients9 quality of life. The transcription factor SPDEF plays a critical role in the regulation of mucus production, and therefore could be a potential target. Here, we present an innovative approach to repress SPDEF expression. Objective: To reduce mucus production by targeted silencing of SPDEF expression with a zinc finger (ZF)-based approach. Methods: Four ZFs were designed to target the SPDEF promoter. They were fused to the Kruppel Associated Box (SKD) repressor domain and ZFs-SKD were lentivirally delivered into the mucoglycoprotein-producing A549 airway epithelial cells. Gene expressions of SPDEF, as well as other mucus-related genes MUC5AC and AGR2 were detected by qRT-PCR. Protein level of SPDEF was investigated by western blot and MUC5AC by immunochemistry staining. Results: Three ZFs-SKD were able to effectively suppress SPDEF expression (up to 96% repression efficiencies) at both mRNA and protein levels. Reduced SPDEF expression resulted in decreased mRNA expression of MUC5AC and AGR2 (both up to 90% repression efficiencies), and also decreased protein expression of MUC5AC. Conclusions: Engineered ZFs-SKD can efficiently reduce SPDEF and consequently, MUC5AC and AGR2 expression. This opens up an innovative avenue to inhibit mucus production by long-lasting epigenetic reprogramming. We currently investigate the effects of ZFs linked to epigenetic editing domains (DNMT3a, G9a) to induce DNA methylation or histone modifications (H3K9me2), in A549 cells and in primary bronchial epithelial cells.