Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor () plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the promoter. All constructs were able to effectively suppress both mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 (), mucin 5AC ()]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%–70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression.

DNA methylation is an essential epigenetic mark, strongly associated with gene expression regulation. Aberrant DNA methylation patterns underlie various diseases and efforts to intervene with DNA methylation signatures are of great clinical interest. Technological developments to target writers or erasers of DNA methylation to specific genomic loci by epigenetic editing resulted in successful gene expression modulation, also in in vivo models.Application of epigenetic editing in human health could have a huge impact, but clinical translation is still challenging. Despite successes for a wide variety of genes, not all genes mitotically maintain their (de)methylation signatures after editing, and reprogramming requires further understanding of chromatin context-dependency. In addition, difficulties of current delivery systems and off-target effects are hurdles to be tackled. The present review describes findings towards effective and sustained DNA (de)methylation by epigenetic editing and discusses the need for multi-effector approaches to achieve highly efficient long-lasting reprogramming.Fabian M. Cortés-Mancera and Federica Sarno contributed equally.