Epigenetic editor therapeutics

Epigenter, Llc, Providence RI

Investigators

Abstract

Abstract. Epigenetic editing modulates the epigenetic code (e.g. cytosine methylation pattern) in key regulatory areas (promoter) of a gene without affecting the patient’s coding DNA sequence, and allows long- lasting modulation of a target gene’s expression while maintaining physiologically appropriate expression range and intrinsic regulatory context. Targeted removal of methyl marks can specifically enhance a gene’s induction by physiological signals or reactivate expression in a cell type that does not typically express it. By coupling DNA demethylase moieties to a sequence specific DNA binding domain (DBD) in a chimeric fusion protein, we were among the first to develop gene-specific epigenetic editors and have achieved localized demethylation of target promoters and specific enhancement of target gene responsiveness. However, this cutting-edge technology has so far remained largely an in vitro research tool. Bringing the technology to the clinic as a new therapeutic strategy is limited by the need for transgenesis to deliver the construct encoding the epigenetic editor into target cells, which is cumbersome and has safety challenges in vivo and causes mechanistic problems in vitro. We propose to eliminate the need for transgene delivery by expressing our epigenetic editors as recombinant, self-delivering proteins which are spontaneously taken up by cells on contact and transferred to the nucleus where they specifically demethylate methylcytosines in the targeted promoter. In preliminary work we have made two classes of epigenetic editors with different practical advantages and weaknesses. In one, the demethylase is fused to an artificial zinc finger protein domain (ZFA), which is spontaneously internalized and binds the chosen DNA sequences. In the other, the demethylase is fused to nuclease-null dCas9 and supercharged peptide; this system will use guide RNAs for binding. Furthermore, instead of a single demethylase, our system employs both Tet1 and TDG, which catalyze separate steps in the demethylation reaction. We propose that these proteins can be delivered directly to tissues as a novel class of biologics that potentiates or enables expression of the target genes, a claim supported by preliminary data in the lung. In this project we will perform feasibility and proof-of-concept studies. We formulated the Aims so as to enable the advance from basic research to a therapeutic design and to support commercialization. In Aim 1 we compare and characterize the in vivo effects of recombinant protein epigenetic editor ‘biologics’ based on ZFA vs. dCAS9 domains. Aim 2 establishes the requirement and compares the effectiveness of two demethylase moieties: TDG and Tet. All constructs target a prototype gene Cxc11 known protective against fibrosis. An alternative target is inducible nitric oxide synthase which, in prior studies, is also upregulatable by epigenetic editing resulting in production of NO that has anti-bacterial effect. Thus targeted demethylation can enable re- activation of intrinsic genes and confers novel properties to cells where these genes are normally silenced.