557: Antisense oligonucleotides modulate nonsense-mediated decay and translation termination pathways to restore expression and function of CFTR harboring nonsense mutations

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

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A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion

Nature Communications ◽

10.1038/s41467-021-24575-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jyoti Sharma ◽

Ming Du ◽

Eric Wong ◽

Venkateshwar Mutyam ◽

Yao Li ◽

...

Keyword(s):

Protein Function ◽

Translation Termination ◽

Nonsense Suppression ◽

Nonsense Mutations ◽

Bronchial Epithelial ◽

Premature Termination Codons ◽

Translational Readthrough ◽

Nonsense Mediated Mrna Decay ◽

Promising Treatment ◽

And Function

AbstractPremature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

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The first identified heterozygous nonsense mutations in podocalyxin offer new perspectives on the biology of podocytopathies

Clinical Science ◽

10.1042/cs20181067 ◽

2019 ◽

Vol 133 (3) ◽

pp. 443-447 ◽

Cited By ~ 2

Author(s):

Ido Refaeli ◽

Michael R. Hughes ◽

Kelly M. McNagny

Keyword(s):

Turning Point ◽

Autosomal Dominant ◽

Adult Onset ◽

Loss Of Function ◽

Nonsense Mutations ◽

Cytoskeletal Organization ◽

Nonsense Mediated Decay ◽

Large Pool ◽

Experimental Findings ◽

And Function

Abstract In the last two decades, our understanding of the genetic underpinnings of inherited podocytopathies has advanced immensely. By sequencing the genomes of a large pool of families affected by focal segmental glomerulosclerosis (FSGS), researchers have identified a common theme: familial podocytopathies are frequently caused by genes selectively expressed in podocytes. Podocalyxin is a podocyte-specific surface sialomucin that has long been known to play important roles in podocyte morphogenesis and function. Few studies, however, have shown a conclusive link between mutations in the gene and FSGS complemented by functional evidence. In a fascinating new paper published in Clinical Science, Lin et al. identify two unrelated pedigrees in which dominant loss-of-function mutations in PODXL lead to adult-onset FSGS. Nonsense-mediated decay of the mutated PODXL transcripts leads to protein insufficiency, which in turn cause podocyte dysfunction through defects in motility and cytoskeletal organization. This is the first study to date that demonstrates, mechanistically, how autosomal dominant mutations in podocalyxin can lead to FSGS and renal insufficiency. Here, we summarize the experimental findings of this manuscript and propose, perhaps, a more controversial hypothesis: down-regulation of podocalyxin protein expression from podocytes is a critical turning point in the progression of most podocytopathies and may be mechanistically relevant to glomerulopathies in which podocyte damage is not necessarily induced by genetic lesions.

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Identification of the Genes Encoding the Cytosolic Translation Release Factors from Podospora anserina and Analysis of Their Role During the Life Cycle

Genetics ◽

10.1093/genetics/149.4.1763 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1763-1775 ◽

Cited By ~ 2

Author(s):

Bénédicte Gagny ◽

Philippe Silar

Keyword(s):

Life Span ◽

Translation Termination ◽

Podospora Anserina ◽

Terminal Region ◽

Terminal Portion ◽

Nonsense Mutations ◽

Translation Fidelity ◽

Reproductive Impairment ◽

Genes Encoding ◽

Nonsense Suppressor

Abstract In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity. We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively. Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span. Deletion of su1, coding for the P. anserina eRF3, is lethal. Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype. P. anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one. Deletion of the N-terminal region of P. anserina eRF3 does not cause any vegetative alteration; especially life span is not changed. However, it promotes a reproductive impairment. Contrary to what happens in S. cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype. Genetic analysis suggests that this domain of eRF3 acts in P. anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

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Faculty Opinions recommendation of Expression of the eRF1 translation termination factor is controlled by an autoregulatory circuit involving readthrough and nonsense-mediated decay in plants.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727180580.793553767 ◽

2018 ◽

Author(s):

Leslie Sieburth

Keyword(s):

Translation Termination ◽

Termination Factor ◽

Nonsense Mediated Decay ◽

Translation Termination Factor

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The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6655 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6655-6662 ◽

Cited By ~ 13

Author(s):

N Yamauchi ◽

A A Kiessling ◽

G M Cooper

Keyword(s):

Monoclonal Antibody ◽

Signaling Pathway ◽

Antisense Oligonucleotides ◽

Subsequent Development ◽

Inhibitory Effect ◽

Mouse Embryos ◽

Dominant Negative ◽

Cell Stage ◽

Cell Embryo ◽

And Function

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

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Screening Readthrough Compounds to Suppress Nonsense Mutations: Possible Application to β-Thalassemia

Journal of Clinical Medicine ◽

10.3390/jcm9020289 ◽

2020 ◽

Vol 9 (2) ◽

pp. 289 ◽

Cited By ~ 3

Author(s):

Monica Borgatti ◽

Emiliano Altamura ◽

Francesca Salvatori ◽

Elisabetta D’Aversa ◽

Nicola Altamura

Keyword(s):

Genetic Disease ◽

Chelation Therapy ◽

Translation Termination ◽

Premature Termination Codon ◽

Nonsense Suppression ◽

Current Status ◽

Nonsense Mutations ◽

Nonsense Mediated Mrna Decay ◽

Mrna Destabilization ◽

Premature Translation Termination

Several types of thalassemia (including β039-thalassemia) are caused by nonsense mutations in genes controlling globin production, leading to premature translation termination and mRNA destabilization mediated by the nonsense mediated mRNA decay. Drugs (for instance, aminoglycosides) can be designed to suppress premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to cure this genetic disease. In the present review, we first summarize the principle and current status of the chemical relief for the expression of functional proteins from genes otherwise unfruitful for the presence of nonsense mutations. Second, we compare data available on readthrough molecules for β0-thalassemia. The examples reported in the review strongly suggest that ribosomal readthrough should be considered as a therapeutic approach for the treatment of β0-thalassemia caused by nonsense mutations. Concluding, the discovery of molecules, exhibiting the property of inducing β-globin, such as readthrough compounds, is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.

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The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.3515 ◽

2011 ◽

Vol 2 (1) ◽

pp. 12 ◽

Cited By ~ 4

Author(s):

Ann Brasey ◽

Raouf Igue ◽

Loubna Djemame ◽

Serge Séguin ◽

Paolo Renzi ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Morphological Changes ◽

Lower Airway ◽

Functional Responses ◽

Phagocytic Capacity ◽

Proinflammatory Mediators ◽

Functional Consequences ◽

And Function

Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.

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