Nonsense-Mediated mRNA Decay and Cystic Fibrosis

Linde, Liat; Kerem, Batsheva

doi:10.1007/978-1-61779-117-8_10

Cited by 12 publications

(5 citation statements)

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“…It is still not clear whether the responses to protein translation correctors might be influenced by an individual capability of ensuring a proper drug mediated read‐through of the mutation by the translation machinery. A possible explanation of the absence of PTC124 effects in four of six cases may be an absence of substrate due to an increased degradation of mRNA containing a premature stop codon, as discussed by Linde and Kerem . Being the patients heterozygous for a nonsense mutation, another possible influence might derive from the presence of the second mutation that might impact on the capability to respond to the treatment.…”

Section: Discussionmentioning

confidence: 88%

Detection of CFTR protein in human leukocytes by flow cytometry

et al. 2014

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Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 lL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels.

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Section: Discussionmentioning

confidence: 88%

Detection of CFTR protein in human leukocytes by flow cytometry

et al. 2014

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“…First, ataluren may not be effective in treating these disorders. However, the numerous preclinical models showing efficacy in other disorders 17,18 make this unlikely if the drug reached affected neurons at therapeutic levels. The drug may not be effective in central nervous system disorders due to limited or erratic crossing of the blood-brain barrier.…”

Section: Discussionmentioning

confidence: 99%

Ataluren for drug‐resistant epilepsy in nonsense variant‐mediated Dravet syndrome and CDKL5 deficiency disorder

Devinsky¹,

King²,

Bluvstein³

et al. 2021

Ann Clin Transl Neurol

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Objective Ataluren is a compound that reads through premature stop codons and increases protein expression by increasing translation without modifying transcription or mRNA stability. We investigated the safety and efficacy of ataluren in children with nonsense variants causing Dravet Syndrome (DS) and CDKL5 Deficiency Syndrome (CDD). Methods This single‐center double‐blind, placebo‐controlled crossover trial randomized subjects to receive ataluren or placebo for 12 weeks (period 1), a 4‐week washout, then another 12‐week treatment (period 2). The primary outcome was ataluren’s safety profile. The secondary outcome measures were (1) changes in convulsive and/or drop seizure frequency and (2) changes in minor seizure types during ataluren treatment compared to placebo. Exploratory objectives assessed changes in cognitive, motor, and behavioral function as well as quality of life during ataluren therapy. Results We enrolled seven subjects with DS and eight subjects with CDD. Three treatment‐related adverse events (AE) occurred during the blinded phases. Two subjects withdrew due to AE. Ataluren was not effective in reducing seizure frequency or improving cognitive, motor, or behavioral function or quality of life in subjects with either DS or CDD due to nonsense variants. Limitations included a small sample size and 12‐week treatment phase, possibly too short to identify a disease‐modifying effect. Significance There was no difference between ataluren and placebo; ataluren is not an effective therapy for seizures or other disorders in children with DS or CDD due to nonsense variants. There were no drug‐related serious AE during the double‐blind period, consistent with ataluren’s favorable safety profile in larger studies. (Funded by Epilepsy Foundation, Dravet Syndrome Foundation, Finding A Cure for Seizures and Epilepsy and PTC Therapeutics, Inc.; ClinicalTrials.gov number, NCT02758626).

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“…Importantly, G542X knockin rats exhibited sensitivity to NMD (Figure 5), a pathway highly relevant to the rescue of nonsense mutations but distinct from transgenic mouse models which do not exhibit this property (Du et al, 2006). CFTR transgenic mice express human CFTR cDNA containing the G542X mutation, but this does not result in intron splicing that triggers NMD when a PTC is encountered during the pioneer round of translation (Maquat, 2004;Du et al, 2006); this substantially reduces the predictive capacity of transgenic mice since humans with CFTR nonsense mutations exhibit NMD sufficient to reduce transcript levels to 20-40% of normal (Sharma et al, 2018;Clarke et al, 2019) and NMD is also known to alter drug response to pharmacological therapy directed against nonsense mutations (Linde and Kerem, 2011;Sharma et al, 2020). Further, the human CFTR cDNA in G542X mice is driven by a rat Fatty Acid Binding Protein (FABP) promoter that results in high levels of intestinalspecific expression, obviating the effects of low transcript levels and limiting tissue assessments to the intestine where it is expressed (Du et al, 2006).…”

Section: Figure 8 |mentioning

confidence: 99%

A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay

et al. 2020

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Nonsense mutations that lead to the insertion of a premature termination codon (PTC) in the cystic fibrosis transmembrane conductance regulator (CFTR) transcript affect 11% of patients with cystic fibrosis (CF) worldwide and are associated with severe disease phenotype. While CF rat models have contributed significantly to our understanding of CF disease pathogenesis, there are currently no rat models available for studying CF nonsense mutations. Here we created and characterized the first homozygous CF rat model that bears the CFTR G542X nonsense mutation in the endogenous locus using CRISPR/Cas9 gene editing. In addition to displaying severe CF manifestations and developmental defects such as reduced growth, abnormal tooth enamel, and intestinal obstruction, CFTR G542X knockin rats demonstrated an absence of CFTR function in tracheal and intestinal sections as assessed by nasal potential difference and transepithelial short-circuit current measurements. Reduced CFTR mRNA levels in the model further suggested sensitivity to nonsense-mediated decay, a pathway elicited by the presence of PTCs that degrades the PTC-bearing transcripts and thus further diminishes the level of CFTR protein. Although functional restoration of CFTR was observed in G542X rat tracheal epithelial cells in response to single readthrough agent therapy, therapeutic efficacy was not observed in G542X knockin rats in vivo. The G542X rat model provides an invaluable tool for the identification and in vivo validation of potential therapies for CFTR nonsense mutations.

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Nonsense-Mediated mRNA Decay and Cystic Fibrosis

Cited by 12 publications

References 10 publications

Detection of CFTR protein in human leukocytes by flow cytometry

Detection of CFTR protein in human leukocytes by flow cytometry

Ataluren for drug‐resistant epilepsy in nonsense variant‐mediated Dravet syndrome and CDKL5 deficiency disorder

A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay

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