Search

Background: Nonsense mutations are at the origin of many cancers and inherited genetic diseases. The consequence of nonsense mutations is often the absence of mutant gene expression due to the activation of an mRNA surveillance mechanism called nonsense-mediated mRNA decay (NMD). Strategies to rescue the expression of nonsense-containing mRNAs have been developed such as NMD inhibition or nonsense mutation readthrough., Methods: Using a dedicated screening system, we sought molecules capable to block NMD. Additionally, 3 cell lines derived from patient cells and harboring a nonsense mutation were used to study the effect of the selected molecule on the level of nonsense-containing mRNAs and the synthesis of proteins from these mutant mRNAs., Results: We demonstrate here that amlexanox, a drug used for decades, not only induces an increase in nonsense-containing mRNAs amount in treated cells, but also leads to the synthesis of the full-length protein in an efficient manner. We also demonstrated that these full length proteins are functional., Conclusions: As a result of this dual activity, amlexanox may be useful as a therapeutic approach for diseases caused by nonsense mutations.

In this paper I review the results of the treatments directed to modify the mRNA of dystrophin with the goal of converting the severe Duchenne type to the milder Becker muscular dystrophy. Antisense oligomers potential to modify Duchenne muscular dystrophy (DMD) gene expression and therapeutic strategies to induce ribosomal read-through of nonsense mutations (PTC124) are described. They are an important advance in the treatment of DMD, so far unspecific. Significant expression of new dystrophin is observed in biopsies of peripheral muscle, although the functional improvement is not so encouraging. New modification of chemistries are expected to improve the liberation, broad distribution in muscles, as well as their efficacy and safety enough to allow a positive chronic treatment of DMD.

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.

Effective cancer pain management requires multidisciplinary approaches for multimodal analgesia. Although opioids have been the cornerstone, developments such as regional anesthesia and interventional pain techniques, complementary and alternative medicine, and new pharmaceuticals also have shown promise to relieve cancer pain. This overview of relevant clinical efforts and the modern day state of the science will afford a better understanding of pain mechanisms and multimodal approaches beneficial in optimizing analgesia for cancer patients., (Copyright © 2012 Wiley Periodicals, Inc.)

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome.

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the -1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable., Competing Interests: The authors have declared that no competing interests exist.

Suppression therapy is a treatment strategy for genetic diseases caused by nonsense mutations. This therapeutic approach utilizes pharmacological agents that suppress translation termination at in-frame premature termination codons (PTCs) to restore translation of a full-length, functional polypeptide. The efficiency of various classes of compounds to suppress PTCs in mammalian cells is discussed along with the current limitations of this therapy. We also elaborate on approaches to improve the efficiency of suppression that include methods to enhance the effectiveness of current suppression drugs and the design or discovery of new, more effective suppression agents. Finally, we discuss the role of nonsense-mediated mRNA decay (NMD) in limiting the effectiveness of suppression therapy, and describe tactics that may allow the efficiency of NMD to be modulated in order to enhance suppression therapy., (Copyright © 2011 John Wiley & Sons, Ltd.)

Duchenne muscular dystrophy is a devastating muscular dystrophy of childhood. Mutations in the dystrophin gene destroy the link between the internal muscle filaments and the extracellular matrix, resulting in severe muscle weakness and progressive muscle wasting. There is currently no cure and, whilst palliative treatment has improved, affected boys are normally confined to a wheelchair by 12 years of age and die from respiratory or cardiac complications in their twenties or thirties. Therapies currently being developed include mutation-specific treatments, DNA- and cell-based therapies, and drugs which aim to modulate cellular pathways or gene expression. This review aims to provide an overview of the different therapeutic approaches aimed at reconstructing the dystrophin-associated protein complex, including restoration of dystrophin expression and upregulation of the functional homologue, utrophin.

Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies., (Copyright © 2011 Wiley-Liss, Inc.)

We investigated the therapeutic potential of the premature termination codon (PTC) readthrough-inducing drug PTC124 in treating the retinal phenotype of Usher syndrome, caused by a nonsense mutation in the USH1C gene. Applications in cell culture, organotypic retina cultures, and mice in vivo revealed significant readthrough and the recovery of protein function. In comparison with other readthrough drugs, namely the clinically approved readthrough-inducing aminoglycoside gentamicin, PTC124 exhibits significant better retinal biocompatibility. Its high readthrough efficiency in combination with excellent biocompatibility makes PTC124 a promising therapeutic agent for PTCs in USH1C, as well as other ocular and nonocular genetic diseases.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by a loss of the dystrophin protein. Control of dystrophin mRNA splicing to convert severe DMD to a milder phenotype is attracting much attention. Here we report a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene. Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein is produced in the patient cells. An analysis of the mRNA reveals that the mutation promotes exon skipping and restores the open reading frame of dystrophin. Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer. Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promotes the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increases the production of the dystrophin protein in the patient's cells.

Approximately one-third of the alleles causing genetic diseases carry premature termination codons (PTCs). Therapeutic approaches for mutations generating in-frame PTCs are aimed at promoting translational readthrough of the PTC, to enable the synthesis and expression of full-length functional proteins. Interestingly, readthrough studies in tissue culture cells, mouse models, and clinical trials revealed a wide variability in the response to the readthrough treatments. The molecular basis for this variability includes the identity of the PTC and its sequence context, the chemical composition of the readthrough drug, and, as we showed recently, the level of PTC-bearing transcripts. One post-transcriptional mechanism that specifically regulates the level of PTC-bearing transcripts is nonsense-mediated mRNA decay (NMD). We have previously shown a role for NMD in regulating the response of CF patients carrying CFTR PTCs to readthrough treatment. Here we describe all the protocols for analyzing CFTR nonsense transcript levels and for investigating the role of NMD in the response to readthrough treatment. This includes inhibition of the NMD mechanism, quantification of CFTR nonsense transcripts and physiologic NMD substrates, and analysis of the CFTR function.

Purpose: The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. USH is clinically and genetically heterogeneous, assigned to three clinical types. The most severe type is USH1, characterized by profound inner ear defects and retinitis pigmentosa. Thus far, no effective treatment for the ophthalmic component of USH exists. The p.R31X nonsense mutation in USH1C leads to a disease causing premature termination of gene translation. Here, we investigated the capability of the novel synthetic aminoglycoside NB30 for the translational read-through of the USH1C-p.R31X nonsense mutation as a retinal therapy option., Methods: Read-through of p.R31X by three commercial, clinically applied aminoglycosides and the synthetic derivative NB30 was validated in vitro, in cell culture, and in retinal explants. Restoration of harmonin functions was monitored in GST pull-downs (scaffold function) and by F-actin bundling analysis in HEK293T cells. Biocompatibility of aminoglycosides was determined in retinal explants by TUNEL assays., Results: In vitro translation and analyses of transfected HEK293T cells revealed a dose-dependent read-through by all aminoglycosides. In addition, gentamicin, paromomycin, and NB30 induced read-through of p.R31X in mouse retinal explants. The read-through of p.R31X restored harmonin protein function. In contrast to all commercial aminoglycosides NB30 showed good biocompatibility., Conclusions: Commercial aminoglycosides and NB30 induced significant read-through of the USH1C-p.R31X nonsense mutation. However, the observed read-through efficiency, along with its significantly reduced toxicity and good biocompatibility, indicate that the novel derivate NB30 represents a better choice than commercial aminoglycosides in a read-through therapy of USH1C and other ocular diseases.

Rationale: Nonsense (premature stop codon) mutations in mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) in approximately 10% of patients. Ataluren (PTC124) is an oral drug that permits ribosomes to readthrough premature stop codons in mRNA to produce functional protein., Objectives: To evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF., Methods: Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles., Measurements and Main Results: The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport. Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults., Conclusions: In children with nonsense mutation CF, ataluren can induce functional CFTR production and is well tolerated.

Nucleotide changes within an exon can alter the trinucleotide normally encoding a particular amino acid, such that a new ''stop'' signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to nonsense-mediated decay of the transcript and lack of production of a normal full-length protein. Such premature termination codon mutations occur in an estimated 10% to 15% of many genetically based disorders, including Duchenne/Becker muscular dystrophy. Therapeutic strategies have been developed to induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein. Small-molecule drugs (aminoglycosides and ataluren [PTC124]) have been developed and are in clinical testing in patients with nonsense mutations within the dystrophin gene. Use of nonsense mutation suppression in Duchenne/Becker muscular dystrophy may offer the prospect of targeting the specific mutation causing the disease and correcting the fundamental pathophysiology.

Rett syndrome is a pediatric neurological condition that affects primarily girls. Approximately 30% of Rett syndrome cases arise from point mutations that introduce a premature stop codon into the MECP2 gene. Several studies have now shown that certain aminoglycosides can facilitate read-through of some types of nonsense mutations in a context-dependent manner and allow the generation of a full-length protein. It remains mostly unclear whether different nonsense mutations of MECP2 will be responsive to aminoglycoside treatment. In this study, we tested whether the common premature terminating mutations of MECP2 seen in Rett syndrome cases can be partially suppressed by aminoglycoside administration. Our results show that aminoglycosides allow different mutant forms of MECP2 to be overcome in transiently transfected HEK293 cells, but with differing levels of efficiency. In addition, we also show that aminoglycosides increased the prevalence of full-length MeCP2 protein in a dose-dependent manner in a lymphocyte cell line derived from a Rett syndrome girl with the R255X mutation. This study helps to establish the "proof of principle" that some nonsense mutations causing Rett syndrome can be at least partially suppressed by drug treatment., ((c) 2010 Wiley-Liss, Inc.)

New pseudo-di- and pseudo-trisaccharide derivatives of the aminoglycoside drug G418 were designed, synthesized and their ability to readthrough nonsense mutations was examined in both in vitro and ex vivo systems, along with the toxicity tests. Two novel lead structures, NB74 and NB84, exhibiting significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin, were discovered. The superiority of new leads was demonstrated in six different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome and Hurler syndrome.

Adenomatous polyposis coli (APC) is a multifunctional tumour suppressor protein that negatively regulates the Wnt signalling pathway. The APC gene is ubiquitously expressed in tissues and organs, including the large intestine and central nervous system. The majority of patients with sporadic and hereditary colorectal cancer have mutations in the gene encoding APC. Approximately 30% of these mutations are single nucleotide changes that result in premature stop codons (nonsense mutations). A potential therapeutic approach for treatment of this subset of patients is the use of aminoglycosides and macrolides that induce nonsense mutation read-through and restore levels of full-length protein. We have used reporter plasmids and colorectal cancer cell lines to demonstrate that several aminoglycosides and tylosin, a member of the macrolide family, induced read-through of nonsense mutations in the APC gene. In xenograft experiments and in the Apc(Min/+) mouse model, these compounds ameliorated the tumorigenic clinical symptoms caused by nonsense mutations in the APC gene.

To induce the readthrough of premature termination codons, aminoglycoside antibiotics such as gentamicin have attracted interest as potential therapeutic agents for diseases caused by nonsense mutations. The transdermal delivery of gentamicin is considered unfeasible because of its low permeability through the dermis. However, if the skin permeability of gentamicin could be improved, it would allow topical application without the need for systemic delivery. In this report, we demonstrated that the skin permeability of gentamicin increased with the use of a thioglycolate-based depilatory agent. After transdermal administration, the readthrough activity in skeletal muscle, as determined using a lacZ/luc reporter system, was found to be equivalent to systemic administration when measured in transgenic mice. Transdermally applied gentamicin was detected by liquid chromatography-tandem mass spectrometry in the muscles and sera of mice only after depilatory agent-treatment. In addition, expansion of the intercellular gaps in the basal and prickle-cell layers was observed by electron microscopy only in the depilatory agent-treated mice. Depilatory agent-treatment may be useful for the topical delivery of readthough-inducing drugs for the rescue of nonsense mutation-mediated genetic disorders. This finding may also be applicable for the transdermal delivery of other pharmacologically active molecules.

Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z'-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency.

Nonsense mutations promote premature translational termination and represent the underlying cause of a large number of human genetic diseases. The aminoglycoside antibiotic gentamicin has the ability to allow the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, severe toxic side effects along with the reduced suppression efficiency at subtoxic doses limit the use of gentamicin for suppression therapy. We describe here the first systematic development of the novel aminoglycoside 2 (NB54) exhibiting superior in vitro readthrough efficiency to that of gentamicin in seven different DNA fragments derived from mutant genes carrying nonsense mutations representing the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy, and Hurler syndrome. Comparative acute lethal toxicity in mice, cell toxicity, and the assessment of hair cell toxicity in cochlear explants further indicated that 2 exhibits far lower toxicity than that of gentamicin.

Rett Syndrome (RTT) is caused in more than 60% of cases by nonsense mutations in the MECP2 gene. So far, no curative therapy for RTT has become available. In other genetic disorders, it has been shown that aminoglycosides can cause a read-through of nonsense mutations with an efficiency of up to 20%. The aim of this study was to evaluate if this therapeutic concept is applicable to RTT. HeLa cells were transfected with eukaryotic expression vectors carrying mutant alleles of frequently occurring MECP2 nonsense mutations that were N-terminally fused to a FLAG tag. Transfected cells were incubated 24 h in the presence of gentamicin. The expression of full-length protein was analyzed by Western blotting and immunofluorescent cell staining. In the presence of gentamicin a read-through varying between 10 and 21.8% was found, depending on the nucleotide sequence context of the nonsense mutations. The full-length protein was located correctly in the nucleus. We have shown that aminoglycoside-mediated read-through of nonsense mutations in the MECP2 gene can be achieved in vitro with efficiency comparable with that seen in other disorders.

Background: Lamivudine is widely used in patients with chronic hepatitis B virus (HBV) infection. In cirrhotic patients, long-term lamivudine therapy significantly reduced the risk of hepatocellular carcinoma (HCC). However, in a small but substantial portion of patients, HCC still developed despite lamivudine therapy. Prolonged usage of lamivudine led to mutations in the polymerase gene, where concurrent nonsense mutations in the HBV S gene occasionally occurred. The significance of such mutations in hepatocarcinogenesis remains elusive. Here, we aimed to understand the oncogenicity of HBV pre-S/S nonsense mutations identified in patients with HCC that developed after lamivudine therapy., Methods: Of 141 consecutive hepatitis B surface antigen-positive HCC patients, 8 developed HCC after receiving lamivudine therapy. The HBV pre-S/S sequences in their serum and tissue samples were analysed. A sex- and age-matched group of HCC patients who never received lamivudine therapy were included as controls. Site-directed mutagenesis experiments were performed to generate identified pre-S/S nonsense mutations in expression vectors for tumourigenicity analysis., Results: Seven of eight patients in the lamivudine-treated group harboured nonsense mutations in the S gene compared with none in the control group (P<0.001). Site-directed mutagenesis and transient transfection experiments revealed that these mutants could transactivate oncogene promoters. NIH3T3 cells stably expressing sL21*, sW156* and sW172* pre-S/S mutants had increased tumourigenicity in nude mice., Conclusions: HCCs developed in lamivudine-treated patients who frequently carried nonsense mutations in the S gene. Such pre-S/S mutants are potentially oncogenic and might counteract the effect of lamivudine in preventing hepatocarcinogenesis.

Background: In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR., Methods: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380., Findings: Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded., Interpretation: In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

Type 1 Usher syndrome (USH1) is a recessively inherited condition, characterized by profound prelingual deafness, vestibular areflexia, and prepubertal onset of retinitis pigmentosa (RP). While the auditory component of USH1 can be treated by cochlear implants, to date there is no effective treatment for RP. USH1 can be caused by mutations in each of at least six genes. While truncating mutations of these genes cause USH1, some missense mutations of the same genes cause nonsyndromic deafness. These observations suggest that partial or low level activity of the encoded proteins may be sufficient for normal retinal function, although not for normal hearing. In individuals with USH1 due to nonsense mutations, interventions enabling partial translation of a full-length functional protein may delay the onset and/or progression of RP. One such possible therapeutic approach is suppression of nonsense mutations by small molecules such as aminoglycosides. We decided to test this approach as a potential therapy for RP in USH1 patients due to nonsense mutations. We initially focused on nonsense mutations of the PCDH15 gene, underlying USH1F. Here, we show suppression of several PCDH15 nonsense mutations, both in vitro and ex vivo. Suppression was achieved both by commercial aminoglycosides and by NB30, a new aminoglycoside-derivative developed by us. NB30 has reduced cytotoxicity in comparison to commercial aminoglycosides, and thus may be more efficiently used for therapeutic purposes. The research described here has important implications for the development of targeted interventions that are effective for patients with USH1 caused by various nonsense mutations.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Premature termination codons represent a common minority of CFTR mutations, and are caused by base pair substitutions that produce abnormal stop codons in the coding sequence. Select aminoglycosides induce "translational readthrough" of premature stop codons and have been shown to restore full-length functional protein in a number of preclinical and clinical settings. We studied two well-described premature termination codons found in the distal open reading frame of CFTR, W1282X and R1162X, expressed in polarizing and nonpolarizing cells. Our findings indicate that W1282X CFTR-expressing cells demonstrate significantly greater CFTR activity when overexpressed compared with R1162X CFTR cells, even when truncated protein is the predominant form. In addition, our results show that the combination of stimulated expression and stop codon suppression produces additive effects on CFTR-mediated ion transport. These findings provide evidence that W1282X CFTR exhibits membrane localization and retained chloride channel function after enhanced expression, and suggest that patients harboring this mutation may be more susceptible to CFTR rescue.

The treatment of genetic disorders is one of the biggest challenges lying ahead of modern medicine. While major advancements have been made in gene therapy, it is still far from achieving clinical success. However, other potential methods for treating single gene related diseases have also emerged recently. One such approach is the suppression of pathogenic nonsense mutations through inducing translational readthrough of the in-frame premature stop mutation. Aminoglycosides were the first drugs that gave promising results in this respect. This report provides a brief overview on the past, present and potential future of this pharmacogenetic approach.

An estimated one third of all inherited genetic disorders and many forms of cancer are caused by premature (nonsense) termination codons. Aminoglycoside antibiotics are candidate drugs for a large number of such genetic diseases; however, aminoglycosides are toxic, lack specificity and show low efficacy in this application. Because translational termination is an active process, we considered that steric hindrance by antisense sequences could trigger the ribosome's "default mode" of readthrough when positioned near nonsense codons. To test this hypothesis, we performed experiments using plasmids containing a luciferase reporter with amber, ochre and opal nonsense mutations within the luxB gene in Escherichia coli. The nonspecific termination inhibitors gentamicin and paromomycin and six antisense peptide nucleic acids (PNA) spanning the termination region were tested for their potential to suppress the luxB mutation. Gentamicin and paromomycin increased luciferase activity up to 2.5- and 10-fold, respectively. Two of the PNAs increased Lux activity up to 2.5-fold over control levels, with no significant effect on cell growth or mRNA levels. Thus, it is possible to significantly suppress nonsense mutations within target genes using antisense PNAs. The mechanism of suppression likely involves enhanced readthrough, but this requires further investigation. Nonsense termination in human cells may also be susceptible to suppression by antisense agents, providing a new approach to address numerous diseases caused by nonsense mutations.

CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. Mutations can be classified according to the mechanisms by which they disrupt CFTR function. This understanding of the different molecular mechanisms of CFTR dysfunction provides the scientific basis for the development of targeted drugs for mutation-specific therapy of cystic fibrosis (CF). Class I mutations are nonsense mutations that result in the presence of a premature stop codon that leads to the production of unstable mRNA, or the release from the ribosome of a short, truncated protein that is not functional. Aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperones such as sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or adenosine tri-phosphate (ATP) binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively, restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the splicing machinery and generate both aberrantly and correctly spliced transcripts, the levels of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increases of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that had to be reached in order to achieve normal function., (Copyright 2005 Wiley-Liss, Inc.)

Approximately 14% of genetic mutations in patients with ataxia-telangiectsia (A-T) are single-nucleotide changes that result in primary premature termination codons (PTCs), either UAA, UAG, or UGA. The purpose of this study was to explore a potential therapeutic approach for this subset of patients by using aminoglycosides to induce PTC read-through, thereby restoring levels of full-length ATM (A-T mutated) protein. In experiments using a modified in vitro cDNA coupled transcription/translation protein truncation test, 13 A-T cell lines carrying PTC mutations in different contexts exhibited read-through expression of ATM fragments, with three of four aminoglycosides tested. In ex vivo experiments with lymphoblastoid cell lines, we used radiosensitivity, radioresistant DNA synthesis, and irradiation-induced autophosphorylation of ATM Ser-1981 to show that the aminoglycoside-induced full-length ATM protein was functional and corrected, to various extents, the phenotype of A-T cells. These results encourage further testing of other compounds in this class, as well as follow up animal studies. Because some A-T patients with 5-20% of normal levels of ATM protein show slower neurological progression, A-T may prove to be a good model for aminoglycoside-induced read-through therapy.

Purpose of Review: The purpose of this review is to summarize the recent approaches using mutation-specific therapy to correct the genetic defect according to the molecular mechanism by which the mutation causes the defects in cystic fibrosis transmembrane conductance regulator (CFTR). Premature stop mutations (class I mutations) account for 5 to 10% of the total mutant alleles in cystic fibrosis patients, and in certain subpopulations the incidence is much higher., Recent Findings: The aminoglycoside antibiotics can suppress premature termination codons by permitting translation to continue to the normal termination of the transcript. The susceptibility to suppression by aminoglycosides depends on the stop codon itself and on the sequence context surrounding it. In vitro studies in cell lines expressing stop mutations and in mice have shown that aminoglycosides caused a dose-dependent increase in CFTR expression and restored functional CFTR to the apical membrane. Clinical studies also provided evidence that the aminoglycoside gentamicin can suppress these CFTR premature stop mutations in affected patients. A recent double-blind, placebo-controlled, crossover study has demonstrated restoration of CFTR function by topical application of gentamicin to the nasal epithelium of cystic fibrosis patients carrying stop mutations. In 21% of the patients there was a complete normalization of all the electrophysiologic abnormalities caused by the CFTR defect, and in 68% there was restoration of either chloride or sodium transport. Furthermore, immunohistochemical staining to the C-terminal part of the CFTR was demonstrated via peripheral staining for CFTR in scraped nasal epithelial cells of patients carrying stop mutations. Inconsistent results were reported regarding the required level of corrected CFTR that has to be reached to achieve normal function. Achieving CFTR activity of 10 to 35% might be needed to prevent significant pulmonary morbidity., Summary: It is as yet unknown how much corrected mutant CFTR must reach the apical membrane to induce a clinically relevant beneficial effect. The future goal is to maximize the effect of stop-codon supressors on CFTR while minimizing side effects, but further studies must be performed to find a safer compound that may be administered in small children from the time of diagnosis.

Many human diseases are caused by inactivating mutations in specific G-protein-coupled receptors (GPCRs). In about 10% of these cases, a premature stop codon leads to the generation of a truncated, functionally inactive receptor protein. In this study, we tested the hypothesis that such GPCR mutations can be functionally rescued in vitro and in vivo by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature termination codons. As a model system, we studied a mutant V2 vasopressin receptor (AVPR2) containing the inactivating E242X nonsense mutation which mimics human X-linked nephrogenic diabetes insipidus (XNDI) when introduced into mice via gene targeting techniques. Studies with cultured mammalian cells expressing the E242X mutant receptor showed that G418 (geneticin) was by far the most potent aminoglycoside antibiotic capable of suppressing the E242X nonsense codon. Strikingly, G418 treatment increased AVP-mediated cAMP responses in cultured kidney collecting duct cells prepared from E242X mutant mice in vitro, and significantly improved the urine-concentrating ability of E242X mutant mice in vivo. This is the first study demonstrating that G418 (aminoglycosides) can ameliorate the clinical symptoms of a disease-causing premature stop codon in a member of the GPCR superfamily.

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (< 2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Background: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing a premature termination signal cause a deficiency or absence of functional chloride-channel activity. Aminoglycoside antibiotics can suppress premature termination codons, thus permitting translation to continue to the normal end of the transcript. We assessed whether topical administration of gentamicin to the nasal epithelium of patients with cystic fibrosis could result in the expression of functional CFTR channels., Methods: In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the DeltaF508 mutation received two drops containing gentamicin (0.3 percent, or 3 mg per milliliter) or placebo in each nostril three times daily for two consecutive periods of 14 days. Nasal potential difference was measured at base line and after each treatment period. Nasal epithelial cells were obtained before and after gentamicin treatment from patients carrying stop mutations, and the C-terminal of surface CFTR was stained., Results: Gentamicin treatment caused a significant reduction in basal potential difference in the 19 patients carrying stop mutations (from -45+/-8 to -34+/-11 mV, P=0.005) and a significant response to chloride-free isoproterenol solution (from 0+/-3.6 to -5+/-2.7 mV, P<0.001). This effect of gentamicin on nasal potential difference occurred both in patients who were homozygous for stop mutations and in those who were heterozygous, but not in patients who were homozygous for DeltaF508. After gentamicin treatment, a significant increase in peripheral and surface staining for CFTR was observed in the nasal epithelial cells of patients carrying stop mutations., Conclusions: In patients with cystic fibrosis who have premature stop codons, gentamicin can cause translational "read through," resulting in the expression of full-length CFTR protein at the apical cell membrane, and thus can correct the typical electrophysiological abnormalities caused by CFTR dysfunction., (Copyright 2003 Massachusetts Medical Society)

Aim of the study was to investigate whether the administration of gentamicin could restore dystrophin expression in striated muscles of patients with Duchenne muscular dystrophy caused by premature stop codon, as reported in mdx mice. Four Duchenne patients, still ambulant or in wheelchair stage for less than 4 months, selected among those with point mutations resulting in premature stop codons, received two 6-day cycles of gentamicin sulfate, at an interval of 7 weeks, according to the protocol approved by the Ethics Committee of the Second University of Naples. A muscle biopsy was performed after the second cycle of administration; the specimens were analysed by both immuno-histochemistry and Western blotting. Skeletal muscle changes were monitored by dynamic tests and Creatine Kinase values; at the beginning and end of treatment, cardiac and respiratory status was evaluated by electrocardiography, echocardiography, acoustic densitometry and vital capacity. Side-effects such as nephrotoxicity and ototoxicity were also monitored. Three out of four patients, who had the most permissive UGA as stop codon, showed positive results. In one patient, there was a dramatic re-expression of dystrophin by both immuno-histochemistry and Western blot; in two patients, dystrophin positive fibres were seen by the antibody to the rod domain with immuno-histochemistry; the fourth patient, with UAA as stop codon, showed no expression of dystrophin at all. These results suggest that gentamicin is able to recover dystrophin expression in a subset of Duchenne patients with nonsense mutations, raising the possibility of the first pharmacological treatment for muscular dystrophy.

Here we report the effects of gentamicin treatment on cystic fibrosis transmembrane regulator (CFTR) production and function in CF airway cells and patients with CF with premature stop mutations. Using immunocytochemical and functional [6-methoxy-N- (3-sulfopropyl) quinolinium (SPQ)-based] techniques, ex vivo exposure of airway cells from stop mutation CF patients led to the identification of surface-localized CFTR in a dose-dependent fashion. Next, five patients with CF with stop mutations and five CF control subjects were treated with parenteral gentamicin for 1 wk, and underwent repeated in vivo measures of CFTR function (nasal potential difference [PD] measurements and sweat chloride [Cl(-)] testing). During the treatment period, the number of nasal PD readings in the direction of Cl(-) secretion was increased approximately 3-fold in the stop mutation patient group compared with controls (p < 0.001), and four of five stop mutation patients with CF had at least one reading during gentamicin treatment with a Cl(-) secretory response of more than -5 mV (hyperpolarized). A response of this magnitude was not seen in any of the CF control subjects (p < 0.05). In an independent series of experiments designed to test the ability of repeat nasal PDs to detect wild-type CFTR function, evidence of Cl(-) secretion was seen in 88% of control (non-CF) nasal PDs, and 71% were more than -5 mV hyperpolarized. Together, these results suggest that gentamicin treatment can suppress premature stop mutations in airway cells from patients with CF, and produce small increases in CFTR Cl(-) conductance (as measured by the nasal PD) in vivo.

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside-induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside-induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside-induced readthrough in the order C > U > A > or = G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33-base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low-level aminoglycoside-induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed.

Germ-line mutations in the human MSH2 (hMSH2) gene account for about 40% of known defects in kindreds with hereditary nonpolyposis colon cancer. We describe a simple fusion protein assay for detection of hMSH2 nonsense mutations in yeast. Detection of nonsense mutations with this assay is severely compromised in many cases by nonsense-mediated mRNA decay, a physiological process that destabilizes the mutant RNA. Triggering of nonsense-mediated decay requires mRNA scanning by the ribosome to detect the stop codon. We show that treatment of cells with the translation inhibitor puromycin suppresses nonsense-mediated decay and facilitates the detection of nonsense mutations in clinical samples by cDNA sequencing, in vitro protein truncation tests, and the yeast fusion protein assay. Given the prevalence of chain-terminating mutations in human disease genes, puromycin treatment of blood samples should improve the signal-to-noise ratio and hence the sensitivity of many RNA-based diagnostic tests. Paradoxically, the yeast hMSH2::ADE2 fusion protein assay also detects some in-frame mutations, presumably through an effect on the folding of the fusion protein.