Epigenome editing of the CFTR-locus for treatment of cystic fibrosis
| Autor: | Rhianna E. Lee, Ian C. McDowell, Scott H. Randell, Nikita Dalal, Ami M. Kabadi, Leah Machlin, Timothy E. Reddy, Nirav N. Shah, Lauren Drowley |
|---|---|
| Rok vydání: | 2022 |
| Předmět: |
0301 basic medicine
Pulmonary and Respiratory Medicine congenital hereditary and neonatal diseases and abnormalities Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cystic fibrosis Article Epigenome 03 medical and health sciences 0302 clinical medicine CRISPR-Associated Protein 9 Epigenome editing Humans Medicine CRISPR ΔF508 Enhancer Gene Editing Regulation of gene expression Messenger RNA Gene knockdown business.industry respiratory system medicine.disease digestive system diseases respiratory tract diseases Cell biology HEK293 Cells 030104 developmental biology Gene Expression Regulation 030228 respiratory system Pediatrics Perinatology and Child Health CRISPR-Cas Systems business RNA Guide Kinetoplastida |
| Zdroj: | J Cyst Fibros |
| ISSN: | 1569-1993 |
| Popis: | 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 dCas9p300 and dCas9KRAB for its ability to enhance or repress CFTR expression, respectively. Results Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9p300 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, dCas9KRAB-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, dCas9p300 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect