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Journal of Cell Science Apr 2016RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse... (Review)
Review
RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts.
Topics: Codon, Nonsense; Humans; Mutation, Missense; Neoplasms; Protein Isoforms; Proto-Oncogene Proteins p21(ras)
PubMed: 26985062
DOI: 10.1242/jcs.182873 -
Nature Apr 2022Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene...
Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene delivery vehicle for in vivo applications. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.
Topics: Adenoviridae; Animals; Codon, Nonsense; Codon, Terminator; Dependovirus; Genetic Diseases, Inborn; Genetic Therapy; Genetic Vectors; Humans; Mice; Nonsense Mediated mRNA Decay; RNA, Transfer
PubMed: 35322228
DOI: 10.1038/s41586-022-04533-3 -
Journal of Comparative Effectiveness... Feb 2022We investigated the effect of ataluren plus standard of care (SoC) on age at loss of ambulation (LoA) and respiratory decline in patients with nonsense mutation...
We investigated the effect of ataluren plus standard of care (SoC) on age at loss of ambulation (LoA) and respiratory decline in patients with nonsense mutation Duchenne muscular dystrophy (nmDMD) versus patients with DMD on SoC alone. Study 019 was a long-term Phase III study of ataluren safety in nmDMD patients with a history of ataluren exposure. Propensity score matching identified Study 019 and CINRG DNHS patients similar in disease progression predictors. Ataluren plus SoC was associated with a 2.2-year delay in age at LoA (p = 0.0006), and a 3.0-year delay in decline of predicted forced vital capacity to <60% in nonambulatory patients (p = 0.0004), versus SoC. Ataluren plus SoC delays disease progression and benefits ambulatory and nonambulatory patients with nmDMD. : NCT01557400.
Topics: Codon, Nonsense; Humans; Muscular Dystrophy, Duchenne; Oxadiazoles; Walking
PubMed: 34791888
DOI: 10.2217/cer-2021-0196 -
Molecular Cell Jan 2023Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons...
Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.
Topics: Animals; Codon, Nonsense; RNA; Codon, Terminator; Mutation; Protein Biosynthesis; Mammals
PubMed: 36521489
DOI: 10.1016/j.molcel.2022.11.011 -
Journal of Neurology Aug 2023Strategic Targeting of Registries and International Database of Excellence (STRIDE) is an ongoing, international, multicenter registry of real-world ataluren use in...
Safety and effectiveness of ataluren in patients with nonsense mutation DMD in the STRIDE Registry compared with the CINRG Duchenne Natural History Study (2015-2022): 2022 interim analysis.
OBJECTIVE
Strategic Targeting of Registries and International Database of Excellence (STRIDE) is an ongoing, international, multicenter registry of real-world ataluren use in individuals with nonsense mutation Duchenne muscular dystrophy (nmDMD) in clinical practice. This updated interim report (data cut-off: January 31, 2022), describes STRIDE patient characteristics and ataluren safety data, as well as the effectiveness of ataluren plus standard of care (SoC) in STRIDE versus SoC alone in the Cooperative International Neuromuscular Research Group (CINRG) Duchenne Natural History Study (DNHS).
METHODS
Patients are followed up from enrollment for at least 5 years or until study withdrawal. Propensity score matching was performed to identify STRIDE and CINRG DNHS patients who were comparable in established predictors of disease progression.
RESULTS
As of January 31, 2022, 307 patients were enrolled from 14 countries. Mean (standard deviation [SD]) ages at first symptoms and at genetic diagnosis were 2.9 (1.7) years and 4.5 (3.7) years, respectively. Mean (SD) duration of ataluren exposure was 1671 (56.8) days. Ataluren had a favorable safety profile; most treatment-emergent adverse events were mild or moderate and unrelated to ataluren. Kaplan-Meier analyses demonstrated that ataluren plus SoC significantly delayed age at loss of ambulation by 4 years (p < 0.0001) and age at decline to %-predicted forced vital capacity of < 60% and < 50% by 1.8 years (p = 0.0021) and 2.3 years (p = 0.0207), respectively, compared with SoC alone.
CONCLUSION
Long-term, real-world treatment with ataluren plus SoC delays several disease progression milestones in individuals with nmDMD. NCT02369731; registration date: February 24, 2015.
Topics: Humans; Codon, Nonsense; Muscular Dystrophy, Duchenne; Registries; Disease Progression
PubMed: 37115359
DOI: 10.1007/s00415-023-11687-1 -
Nature Jun 2022Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral. Here, to experimentally...
Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar-albeit nonidentical-between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.
Topics: Amino Acid Sequence; Codon, Nonsense; Evolution, Molecular; Genes, Fungal; Genetic Fitness; Mutation Rate; RNA, Fungal; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Selection, Genetic; Silent Mutation
PubMed: 35676473
DOI: 10.1038/s41586-022-04823-w -
Journal of Comparative Effectiveness... Dec 2021Compare efficacies of deflazacort and prednisone/prednisolone in providing clinically meaningful delays in loss of physical milestones in patients with nonsense... (Meta-Analysis)
Meta-Analysis
Compare efficacies of deflazacort and prednisone/prednisolone in providing clinically meaningful delays in loss of physical milestones in patients with nonsense mutation Duchenne muscular dystrophy. Placebo data from Phase IIb (ClinicalTrials.gov Identifier: NCT00592553) and ACT DMD (ClinicalTrials.gov Identifier: NCT01826487) ataluren nonsense mutation Duchenne muscular dystrophy clinical trials were retrospectively combined in meta-analyses (intent-to-treat population; for change from baseline to week 48 in 6-min walk distance [6MWD] and timed function tests). Significant improvements in change in 6-min walk distance with deflazacort versus prednisone/prednisolone (least-squares mean difference 39.54 m [95% CI: 13.799, 65.286; p = 0.0026]). Significant and clinically meaningful improvements in 4-stair climb and 4-stair descend for deflazacort versus prednisone/prednisolone. Deflazacort provides clinically meaningful delays in loss of physical milestones over 48 weeks compared with prednisone/prednisolone for patients with nonsense mutation Duchenne muscular dystrophy.
Topics: Codon, Nonsense; Humans; Muscular Dystrophy, Duchenne; Prednisolone; Prednisone; Pregnenediones; Retrospective Studies
PubMed: 34693725
DOI: 10.2217/cer-2021-0018 -
Cells Jan 2023Up-frameshift protein 1 (UPF1) plays the role of a vital controller for transcripts, ready to react in the event of an incorrect translation mechanism. It is well known... (Review)
Review
Up-frameshift protein 1 (UPF1) plays the role of a vital controller for transcripts, ready to react in the event of an incorrect translation mechanism. It is well known as one of the key elements involved in mRNA decay pathways and participates in transcript and protein quality control in several different aspects. Firstly, UPF1 specifically degrades premature termination codon (PTC)-containing products in a nonsense-mediated mRNA decay (NMD)-coupled manner. Additionally, UPF1 can potentially act as an E3 ligase and degrade target proteins independently from mRNA decay pathways. Thus, UPF1 protects cells against the accumulation of misfolded polypeptides. However, this multitasking protein may still hide many of its functions and abilities. In this article, we summarize important discoveries in the context of UPF1, its involvement in various cellular pathways, as well as its structural importance and mutational changes related to the emergence of various pathologies and disease states. Even though the state of knowledge about this protein has significantly increased over the years, there are still many intriguing aspects that remain unresolved.
Topics: Humans; RNA Helicases; Trans-Activators; Nonsense Mediated mRNA Decay; Mutation; Codon, Nonsense
PubMed: 36766761
DOI: 10.3390/cells12030419 -
Wiley Interdisciplinary Reviews. RNA Jul 2021Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single-nucleotide substitution. The generation of a PTC results... (Review)
Review
Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single-nucleotide substitution. The generation of a PTC results in a defective truncated protein and often in severe forms of disease. Because of the exceedingly high prevalence of nonsense-associated diseases and a unifying mechanism, there has been a concerted effort to identify PTC therapeutics. Most clinical trials for PTC therapeutics have been conducted with small molecules that promote PTC read through and incorporation of a near-cognate amino acid. However, there is a need for PTC suppression agents that recode PTCs with the correct amino acid while being applicable to PTC mutations in many different genomic landscapes. With these characteristics, a single therapeutic will be able to treat several disease-causing PTCs. In this review, we will focus on the use of nonsense suppression technologies, in particular, suppressor tRNAs (sup-tRNAs), as possible therapeutics for correcting PTCs. Sup-tRNAs have many attractive qualities as possible therapeutic agents although there are knowledge gaps on their function in mammalian cells and technical hurdles that need to be overcome before their promise is realized. This article is categorized under: RNA Processing > tRNA Processing Translation > Translation Regulation.
Topics: Animals; Codon, Nonsense; Mutation; RNA, Transfer
PubMed: 33567469
DOI: 10.1002/wrna.1641 -
International Journal of Molecular... Nov 2021Nonsense mutations are the result of single nucleotide substitutions in the DNA that change a sense codon (coding for an amino acid) to a nonsense or premature...
Nonsense mutations are the result of single nucleotide substitutions in the DNA that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of the mRNA [...].
Topics: Codon, Nonsense; Genetic Diseases, Inborn; Humans; Mutation; Nonsense Mediated mRNA Decay; Protein Biosynthesis
PubMed: 34769359
DOI: 10.3390/ijms222111933