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Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal for targeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities., Competing Interests: Declaration of interests A.C. is a co-founder and holds stocks of Alia Therapeutics, a genome editing company., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Pharmacological inhibitors of TMEM16A (ANO1), a Ca 2+ -activated Cl - channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCC inh -A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca 2+ ., Experimental Approach: We recorded cytosolic Ca 2+ increase elicited with UTP, ionomycin or IP 3 uncaging., Key Results: Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca 2+ elevation induced by stimuli acting on intracellular Ca 2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCC inh -A01. Acute addition of niclosamide directly increased intracellular Ca 2+ , an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCC inh -A01 did not elevate intracellular Ca 2+ , thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors., Conclusions and Implications: Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca 2+ handling, which may in part preclude their use as TMEM16A research tools., (© 2022 British Pharmacological Society.)

Pharmacological modulators of the Ca 2+ signaling cascade are important research tools and may translate into novel therapeutic strategies for a series of human diseases. We carried out a screening of a maximally diverse chemical library using the Ca 2+ -sensitive Cl - channel TMEM16A as a functional readout. We found compounds that were able to potentiate UTP-dependent TMEM16A activation. Mechanism of action of these compounds was investigated by a panel of assays that looked at intracellular Ca 2+ mobilization triggered by extracellular agonists or by caged-IP 3 photolysis, PIP 2 breakdown by phospholipase C, and ion channel activity on nuclear membrane. One compound appears as a selective potentiator of inositol triphosphate receptor type 1 (ITPR1) with a possible application for some forms of spinocerebellar ataxia. A second compound is instead a potentiator of the P2RY2 purinergic receptor, an activity that could promote fluid secretion in dry eye and chronic obstructive respiratory diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 β-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a β-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition.