Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.

Keywords: ASO delivery; CFTR; Tat-CPP; cystic fibrosis; exosome; lncRNA BGas; transcriptional regulation.

Figure 1

Activation of CFTR Expression by dCas9/VPR (A) Real-time qPCR of CFTR expression after screening seven gRNAs (g1–g7) directed to the CFTR promoter (A1, control) using CFPAC cells. (B) Top, chromosome 7 ideogram showing CFTR gene. Bottom, close up snapshot of the UCSC browser of the gRNA (g3) located upstream of CFTR promoter. Position is relative to the transcription start site (TSS). (C and D) CFTR promoter-directed guide RNA (g3) (C) activates CFTR mRNA expression in nasal cells from healthy donor-WT and (D) in nasal cells from a CF patient with F508del mutation. (E and F) CFTR protein overexpression enriches after treatment with dCas9/VPR. CFTR ELISA showing CFTR protein level expression increased after being treated with g3RNA/dcas9-VRP in (E) nasal cells from healthy donor-WT and (F) in nasal cells from a CF patient with F508del mutation. Experiments were performed in triplicate in cells shown with the SEMs and p values from a paired two-sided t test, *p = 0.001.

Figure 2

BGas Repression Results in Increased Expression of CFTR (A) Screen panel of ten gapmers. (B) Top, diagram of CFTR and BGas showing gapmer targeting BGas intron 1, bottom. (C) Directional RT-PCR (inverted picture of an end-point PCR) of BGas and (D) relative densitometric analysis of the individual band in nasal cells from a CF patient with F508del. (E and F) Gapmer-Tat CPP increased CFTR expression in (E) nasal cells from healthy donor-WT and (F) in nasal cells from a CF patient with F508del mutation. CFTR expression was determined by qRT-PCR. (G and H) CFTR ELISA showing CFTR protein level expression increased after being treated with BGas-gapmer in (G) nasal cells from healthy donor-WT and (H) nasal cells from a CF patient with F508del mutation. For directional RT-real-time qPCR (C) and ELISA (G and H), experiments were performed in triplicate in cells shown with the SEMs and p values from a paired two-sided t test, *p = 0.001, **p < 0.05.

Figure 3

Exosome-Mediated Delivery of Gapmer Enters Human Nasal Cells and Increases Expression of CFTR (A) Transmission electron microscopy (TEM) micrographs of exosomes isolated from the culture medium of A549 cells. Exosomes were measured by using a Nanosight NS-300 system in the supernatant from culture cells. The histogram represents particle size distribution. (B) Confocal analysis of uptake of gapmer10-Cy5 electroporated exosomes into nasal cells. (C) Real-time qPCR of CFTR mRNA levels in nasal cells from healthy donor-WT and (D) nasal cells from a CF patient with F508del mutation treated with exosome package gapmer 10 (G10) or scramble (Scr). (E and F) CFTR ELISA was carried out for CFTR protein expression in human nasal cells treated with exosomes carrying BGas-gapmer 10 (G10) or control scramble (Scr) from (E) healthy donor-WT and (F) nasal cells from a CF patient with F508del mutation. Scale bar, 20 μm. Experiments were performed in triplicate in cells shown with the SEMs and p values from a paired two-sided t test, *p = 0.001, **p < 0.05.

Figure 4

CFTR-Mediated Halide Transport in EYFP (A) Schematic diagram depicting the halide assay. (B) Human nasal F508del expressing EYFP. Cells were incubated with 0.3 μM of forskolin, 10 μM amiloride, and 100 μM of niflumic acid. Fluorescence measured in response to exchange of 25 mM of sodium iodide. Fluorescence decrease in human nasal cells, WT (C) and F508del (D), treated with electrostatic particle gapmer-Tat (G10-Tat) or scramble (Scr-Tat). Fluorescence change in nasal cells from healthy donor-WT (E) and nasal cells from a CF patient with F508del mutation (F) treated with no exosomes (NE) or with exosomes packed with gapmer (Exo-G10) or scramble (Exo-Scr). Functional halide assay in nasal cells from healthy donor-WT (G) and nasal cells from a CF patient with F508del mutation (H) treated with no lipid nanoparticles (No-LNP) or with LNPs loaded with gapmer (LNP-G10) or scramble (LNP-Scr). Experiments were performed in triplicate in cells shown with the SEMs and p values from a paired two-sided t test, *p = 0.01, **p $\underset{¯}{\le }$0.05.