Isogenic cell models of cystic fibrosis-causing variants in natively expressing pulmonary epithelial cells

Abstract

Background: Assessment of approved drugs and developmental drug candidates for rare cystic fibrosis (CF)-causing variants of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) requires abundant material from relevant models. Methods: Isogenic cell lines harboring CFTR variants in the native genomic context were created through the development and utilization of a footprint-less, CRISPR/Cas9 gene editing pipeline in 16HBE14o-immortalized bronchial epithelial cells. Results: Isogenic, homozygous cell lines for three CFTR variants (F508del and the two most common CF-causing nonsense variants, G542X and W1282X) were established and characterized. The F508del model recapitulates the known molecular pathology and pharmacology. The two models of nonsense variants (G542X and W1282X) are sensitive to Nonsense Mediated mRNA Decay (NMD) and responsive to reference compounds that inhibit NMD and promote ribosomal readthrough. Conclusions: We present a versatile, efficient gene editing pipeline that can be used to create CFTR variants in the native genomic context and the utilization of this pipeline to create homozygous cell models for the CF-causing variants F508del, G542X, and W1282X. The resulting cell lines provide a virtually unlimited source of material with specific pathogenic mutations that can be used in a variety of assays, including functional assays.

Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope

“…The second most common CFTR nonsense mutations, W1282X 25,26 , is caused by a G>A mutation in exon 23 that produces minimal amount of functional CFTR protein and abolishes its Cl − transport activity 27 . A previous report showed that the human bronchial epithelial cell line (CFF-16HBEge CFTR W1282X), homozygous for W1282X mutation, recapitulates the phenotype of primary W1282X CF cells 25 , making it a good cell model to study the correction of CFTR mutation. SV40 (polyomavirus simian virus 40), which was used to immortalize the 16HBEge cell line's parental 16HBE14o-cells, is integrated into one of the CFTR alleles 28 .…”

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies

“…Transient expression of EMGs in HEK293s cannot account for the effects of chromatin, however stably integrated EMGs in our CFBE cell lines are subject to chromatin-level regulation, albeit at a different genomic locus than endogenous CFTR . While CRISPR/Cas and other methods of gene editing can generate cell lines expressing CFTR variants in the native genomic context [ 64 , 65 ], EMGs provide a more rapid approach for assessing a large number of variants to identify those with unexpected consequences that warrant follow-up studies. Additionally, we show here and in prior work by ourselves and others that our results in EMGs are corroborated by studies in primary cells [ 20 , 35 – 38 , 44 , 46 , 50 , 66 ].…”

Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing

“…However, considering the high number of CFTR variants associated with CF, it is very difficult to cover the entire mutation repertoire in homozygous cellular models. To overcome this limitation, isogenic models for different mutations were created using CRISPR-Cas9 mediated HDR in immortalized bronchial epithelial cells providing valuable experimental tools for CFTR functional assay [95].…”