scholarly journals Efficient Suppression of Endogenous CFTR Nonsense Mutations Using Anticodon Engineered Transfer RNAs

2021 ◽  
Author(s):  
Wooree Ko ◽  
Joseph J. Porter ◽  
Matthew T. Sipple ◽  
Katherine M. Edwards ◽  
John D. Lueck
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Genetic Diseases ◽  
Protein Translation ◽  
Nonsense Mutations ◽  
Channel Function ◽  
Associated Diseases ◽  
Highly Active ◽  
Premature Termination Codons ◽  
Efficient Suppression

Nonsense mutations or premature termination codons (PTCs) comprise ~11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases, however their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542X-, R1162X- and W1282X-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential stand-alone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC containing endogenous genes.

scholarly journals Translational Read-Through Therapy of RPGR Nonsense Mutations

2020 ◽  
Vol 21 (22) ◽  
pp. 8418
Author(s):  
Christine Vössing ◽  
Marta Owczarek-Lipska ◽  
Kerstin Nagel-Wolfrum ◽  
Charlotte Reiff ◽  
Christoph Jüschke ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Western Blot ◽  
Primary Cilium ◽  
Genetic Diseases ◽  
The Novel ◽  
Rt Pcr ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
New Treatment ◽  
First Time

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, TranslamaTM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases.

scholarly journals Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons

2018 ◽  
Vol 4 (1) ◽  
pp. 00080-2017 ◽  
Author(s):  
Iwona Pranke ◽  
Laure Bidou ◽  
Natacha Martin ◽  
Sandra Blanchet ◽  
Aurélie Hatton ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Premature Termination ◽  
Genetic Diseases ◽  
Exon Skipping ◽  
Reporter System ◽  
Drug Induced ◽  
Premature Termination Codons ◽  
Promising Therapeutic Approach ◽  
Dual Reporter ◽  
Cftr Modulators

Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators.First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments.The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient.In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.

scholarly journals 2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases

2021 ◽  
Author(s):  
Laure Bidou ◽  
Olivier Bugaud ◽  
Goulven Merer ◽  
Matthieu Coupet ◽  
Isabelle Hatin ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
High Throughput Screening ◽  
Therapeutic Strategy ◽  
Genetic Diseases ◽  
Medical Applications ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Clinical Potential

Premature termination codons (PTCs) account for 10% to 20% of genetic diseases in humans. The gene inactivation resulting from PTC can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a new reporter cell line and performed high-throughput screening (HTS) to identify potential new readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy. We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of natural stop codons.

scholarly journals Strategies against Nonsense: Oxadiazoles as Translational Readthrough-Inducing Drugs (TRIDs)

2019 ◽  
Vol 20 (13) ◽  
pp. 3329 ◽  
Author(s):  
Ambra Campofelice ◽  
Laura Lentini ◽  
Aldo Di Leonardo ◽  
Raffaella Melfi ◽  
Marco Tutone ◽  
...  
Keyword(s):  
Small Molecules ◽  
Genetic Diseases ◽  
Nonsense Mutations ◽  
New Class ◽  
Premature Termination Codons ◽  
Translational Readthrough ◽  
Action Strategies ◽  
Oxadiazole Derivatives ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs.

scholarly journals CFTR Cooperative Cis-Regulatory Elements in Intestinal Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2599
Author(s):  
Mégane Collobert ◽  
Ozvan Bocher ◽  
Anaïs Le Nabec ◽  
Emmanuelle Génin ◽  
Claude Férec ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Gene Regulation ◽  
Genetic Diseases ◽  
Regulatory Elements ◽  
Cftr Gene ◽  
Intestinal Cells ◽  
Cooperative Effects ◽  
Cis Acting ◽  
Study Techniques

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as “cis-ruptions” have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.

Optimal Genetic Screening for Cystic Fibrosis

2021 ◽  
Author(s):  
Hussein El Hajj ◽  
Douglas R. Bish ◽  
Ebru K. Bish
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Variants ◽  
Genetic Screening ◽  
Genetic Diseases ◽  
The United States ◽  
Published Data ◽  
Decision Support Model ◽  
Screening Process ◽  
Public Health Initiative ◽  
Cystic Fibrosis Screening

Improving Newborn Screening for Genetic Diseases Screening newborns for life-threatening genetic diseases is an important public health initiative. Cystic fibrosis is one of the most prevalent diseases in this context. As part of the cystic fibrosis screening process, all states in the United States use multiple tests, including genetic tests that detect a subset of the more than 300 genetic variants (specific mutations) that cause cystic fibrosis. In “Optimal Genetic Screening for Cystic Fibrosis,” El-Hajj, D.R. Bish, and E.K. Bish develop a decision support model to select which genetic variants to screen for, considering the trade-off between classification accuracy and testing cost, and the technological constraints that limit the number of variants selected. Because variant prevalence rates are highly uncertain, a robust optimization framework is developed. Further, two commonly used cystic fibrosis screening processes are analytically compared, and conditions under which each process dominates are established. A case study based on published data are provided.

scholarly journals Prevalence and impact of oprD mutations in Pseudomonas aeruginosa strains in cystic fibrosis

2019 ◽  
Author(s):  
Laura J. Sherrard ◽  
Bryan A. Wee ◽  
Christine Duplancic ◽  
Kay A. Ramsay ◽  
Keyur A. Dave ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lung Function ◽  
Hazard Rate ◽  
Carbapenem Resistance ◽  
Adverse Outcomes ◽  
Nonsense Mutations ◽  
Allelic Variants ◽  
Novel Variants

ABSTRACTDefective OprD porins contribute to carbapenem resistance and may be important in Pseudomonas aeruginosa adaptation to cystic fibrosis airways. It is unclear whether oprD mutations are fixed in populations of shared strains that are transmitted between patients or whether novel variants arise during infection. We investigated oprD sequences and antimicrobial resistance of two common Australian shared strains, constructed P. aeruginosa mutants with the most common oprD allelic variants and compared characteristics between patients with or without evidence of infection with strains harbouring these variants. Our data show that three independently acquired nonsense mutations arising from a 1-base pair substitution are fixed in strain sub-lineages. These nonsense mutations are likely to contribute to reduced carbapenem susceptibility in the sub-lineages without compromising in vitro fitness. Not only was lung function worse among patients infected with strains harbouring the nonsense mutations than those without, but they also had an increased hazard rate of lung transplantation/death. Our findings further highlight that understanding adaptive changes may help to distinguish patients with greater adverse outcomes despite infection with the same strain.

scholarly journals Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Cristina Lazzarini ◽  
Krupanandan Haranahalli ◽  
Robert Rieger ◽  
Hari Krishna Ananthula ◽  
Pankaj B. Desai ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Fungal Resistance ◽  
Content Type ◽  
Highly Active ◽  
Pharmacokinetic Properties ◽  
Pass Through

ABSTRACTThe incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active againstCryptococcus neoformansin vitroand had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

Complex Genetic Diseases

2016 ◽  
Author(s):  
Michael Snyder
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Disease ◽  
Single Gene ◽  
Genetic Diseases ◽  
Gene Variants ◽  
Tay Sachs Disease ◽  
Causative Effect ◽  
Complex Genetic Diseases

What is a complex genetic disease? Although great strides have been made to identify single gene variants that have a strong causative effect for a particular disease (e.g., CFTR mutations for cystic fibrosis and HEXA mutations for Tay-Sachs disease), the...

scholarly journals Therapy for Cystic Fibrosis Caused by Nonsense Mutations

10.5772/61053 ◽  
2015 ◽  
Author(s):  
Roberto Gambari ◽  
Giulia Breveglieri ◽  
Francesca Salvatori ◽  
Alessia Finotti ◽  
Monica Borgatti
Keyword(s):  
Cystic Fibrosis ◽  
Nonsense Mutations
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