Vector-free intra-airway in vivo epigenetic editing

Targeted, promoter-specific removal of DNA methylation marks is emerging as a promising strategy for experimental and therapeutic regulation of gene expression. Research to date has relied largely on the expression of transgene-encoded epigenetic editor constructs in target cells. While effective for in vitro demonstrations, alternative approaches are needed for greater translatability to humans. Here, we describe the design of recombinant, gene-targeted epigenetic editor proteins that are directly taken up by mouse lung cells following administration in vivo without transgenesis, vectors, or packaging tools. Proteins are targeted to their intended promoter using either dCas9 or artificial zinc finger domains, and thymine-DNA-glycosylase (TDG) and ten-eleven translocation proteins (Tet) catalytic domains mediate specific demethylation. Results demonstrate intranuclear arrival of epigenetic editors in vitro and in mice, local DNA demethylation, and resulting highly gene-specific derepression of the transcriptional response, which confers sensitivity to interferon (IFN) stimulation. Therefore, this study provides proof of principle for vector-free, targeted promoter demethylation in vivo.