Epigenome editing of the CFTR-locus for treatment of cystic fibrosis

Abstract

Background: Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues.

Methods: Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9^p300 and dCas9^KRAB for its ability to enhance or repress CFTR expression, respectively.

Results: Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9^p300 mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9^KRAB-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9^p300 was used to induce elevated levels of CFTR from the endogenous locus in ΔF508/ΔF508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of ΔF508 CFTR mRNA along with VX809 treatment.

Conclusions: CRISPR/dCas9-based epigenome-editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.

Keywords: CFTR; CRISPR/dCas9; Cystic fibrosis; Epigenome-editing; Gene regulation.