Ellen Welch

PTC124 targets genetic disorders caused by nonsense mutations

Nonsense mutations promote premature translational termination and cause anywhere from 5–70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124—a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2–8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

Safety, Tolerability, and Pharmacokinetics of PTC124, a Nonaminoglycoside Nonsense Mutation Suppressor, Following Single‐ and Multiple‐Dose Administration to Healthy Male and Female Adult Volunteers

Nonsense (premature stop codon) mutations are causative in 5% to 15% of patients with monogenetic inherited disorders. PTC124, a 284‐Dalton 1,2,4‐oxadiazole, promotes ribosomal readthrough of premature stop codons in mRNA and offers therapeutic potential for multiple genetic diseases. The authors conducted 2 phase I studies of PTC124 in 62 healthy adult volunteers. The initial, single‐dose study evaluated doses of 3 to 200 mg/kg and assessed fed‐fasting status on pharmacokinetics following a dose of 50 mg/kg. The subsequent multiple‐dose study evaluated doses from 10 to 50 mg/kg/dose twice per day (bid) for up to 14 days. PTC124 administered orally as a liquid suspension was palatable and well tolerated through single doses of 100 mg/kg. At 150 and 200 mg/kg, PTC124 induced mild headache, dizziness, and gastrointestinal events. With repeated doses through 50 mg/kg/dose bid, reversible transaminase elevations <2 times the upper limit of normal were sometimes observed. Immunoblot analyses of peripheral blood mononuclear cell extracts revealed no protein elongation due to nonspecific ribosomal readthrough of normal stop codons. PTC124 plasma concentrations exceeding the 2‐ to 10‐μg/mL values associated with activity in preclinical genetic disease models were safely achieved. No sex‐related differences in pharmacokinetics were seen. No drug accumulation with repeated dosing was apparent. Diurnal variation was observed, with greater PTC124 exposures after evening doses. PTC124 excretion in the urine was <2%. PTC124 pharmacokinetics were described by a 1‐compartment model. Collectively, the data support initiation of phase II studies of PTC124 in patients with nonsense mutation–mediated cystic fibrosis and Duchenne muscular dystrophy.

PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model

Nonsense mutations inactivate gene function and are the underlying cause of a large percentage of the individual cases of many genetic disorders. PTC124 is an orally bioavailable compound that promotes readthrough of premature translation termination codons, suggesting that it may have the potential to treat genetic diseases caused by nonsense mutations. Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr−/− mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Translational readthrough of the premature stop codon was demonstrated in this mouse model in two ways. First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr−/− hCFTR-G542X mice. In addition, functional assays demonstrated that PTC124 treatment restored 24–29% of the average cAMP-stimulated transepithelial chloride currents observed in wild-type mice. These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. In light of its oral bioavailability, safety toxicology profile in animal studies, and efficacy with other nonsense alleles, PTC124 has the potential to be an important therapeutic agent for the treatment of inherited diseases caused by nonsense mutations.

Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial

BACKGROUND Ataluren was developed to restore functional protein production in genetic disorders caused by nonsense mutations, which are the cause of cystic fibrosis in 10% of patients. This trial was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis. METHODS This randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged ≥ 6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV1] ≥ 40% and ≤ 90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV1. The primary endpoint was relative change in percent-predicted FEV1 from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205. FINDINGS Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV1 did not differ significantly between ataluren and placebo at week 48 (-2.5% vs -5.5%; difference 3.0% [95% CI -0.8 to 6.3]; p=0.12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0.77 [95% CI 0.57-1.05]; p=0.0992). However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5.7% difference (95% CI 1.5-10.1) in relative change from baseline in percent-predicted FEV1 between the ataluren and placebo groups at week 48 (-0.7% [-4.0 to 2.1] vs -6.4% [-9.8 to -3.7]; nominal p=0.0082), and fewer pulmonary exacerbations in the ataluern group (1.42 events [0.9-1.9] vs 2.18 events [1.6-2.7]; rate ratio 0.60 [0.42-0.86]; nominal p=0.0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group. INTERPRETATION Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin. FUNDING PTC Therapeutics, Cystic Fibrosis Foundation, US Food and Drug Administrations Office of Orphan Products Development, and the National Institutes of Health.

Ataluren as an Agent for Therapeutic Nonsense Suppression

The interplay of translation and mRNA turnover has helped unveil how the regulation of gene expression is a continuum in which events that occur during the birth of a transcript in the nucleus can have profound effects on subsequent steps in the cytoplasm. Exemplifying this continuum is nonsense-mediated mRNA decay (NMD), the process wherein a premature stop codon affects both translation and mRNA decay. Studies of NMD helped lead us to the therapeutic concept of treating a subset of patients suffering from multiple genetic disorders due to nonsense mutations with a single small-molecule drug that modulates the translation termination process at a premature nonsense codon. Here we review both translation termination and NMD, and our subsequent efforts over the past 15 years that led to the identification, characterization, and clinical testing of ataluren, a new therapeutic with the potential to treat a broad range of genetic disorders due to nonsense mutations.

Nonsense-mediated mRNA decay in yeast

Publisher Summary This chapter describes the mRNA-destabilizing phenomenon as nonsense-mediated mRNA decay, and reviews the cis-acting sequences and trans-acting factors that comprise this pathway in the yeast Saccharomyces cerevisiae. The observation that premature translation termination can promote rapid mRNA decay is just one in a large set of observations that point to an important role for translation in the process of mRNA decay. Additional evidence for this linkage comes from experiments showing that: (1) inhibitors of translation elongation (e.g., cycloheximide) or mutations that inhibit protein synthesis stabilize mRNAs; (2) mRNAs undergoing rapid decay are polysome associated; (3) degradative factors can be polysome associated; (4) instability elements are located in coding regions; (5) the activity of coding-region instability-elements depends on ribosome translocation up to or near the element; and (6) metabolism of the poly(A) tail, a structure involved in translation initiation is a rate-limiting step in the decay of several mRNAs. Although these observations are consistent with the postulated role of ribosomal translocation, the requirement for translation is not likely to be limited to a single step in the turnover process.

Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression.

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (approximately 15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Significance The drug ataluren restores activity to otherwise nonfunctional nonsense alleles, a capability possibly reflecting the insertion of near-cognate aminoacyl tRNAs at premature termination codons during protein synthesis. Because nonsense alleles comprise a significant fraction of all alleles causing inherited disorders, drugs that promote such nonsense codon readthrough have broad therapeutic potential. However, the effectiveness of therapeutic nonsense suppression depends on the nature of the amino acids inserted at each of the three nonsense codons. Here we demonstrate that ataluren does indeed promote insertion of near-cognate tRNAs at nonsense codons, that the latter process yields functional proteins, and that specific codon:anticodon base pairings are critical to this process. These results should enable predictions of better clinical outcomes with therapeutic nonsense suppression. A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

Nonsense suppression activity of PTC124 (ataluren)

Auld et al. (1) suggest that PTC124s nonsense suppression activity may be an indirect consequence of the compounds effects on firefly luciferase (FLuc) enzymatic activity. However, our initial characterization of potential nonsense-suppressing compounds in FLuc assays also utilized independent assays of nonsense suppression in disease-relevant systems, including assays measuring synthesis of full-length protein in mdx myotubes, mdx mice, and Cftr−/− transgenic mice (2, 3). These tests eliminated inactive chemical scaffolds and identified PTC124. Subsequently, PTC124 demonstrated nonsense suppression activity in cystic fibrosis and Duchenne muscular dystrophy patients (2, 4). These independent confirmations in validated suppression assays are to be contrasted with Auld et al.s (1) use of a Renilla luciferase reporter that was an unsuitable and misinterpreted gauge of nonsense suppression because it failed to respond appreciably to the established nonsense-suppressing aminoglycoside, G418.

11 Translation Termination: It’s Not the End of the Story

The protein synthesis apparatus must carry out translation initiation, elongation, and termination to synthesize a complete protein product. Each step of this process has evolved to occur with great accuracy and can be utilized to regulate gene expression. Until relatively recently, however, translation termination was the least investigated and understood aspect of the translation process. Recent results, however, suggest that translation termination also can be used to modulate gene expression. The fact that sequences in the 3′-untranslated regions (3′UTRs) of mRNAs bind to RNA-binding proteins and can dramatically affect the translation initiation process suggests that steps which occur at translation termination may strongly affect subsequent events in the translation process. It is conceivable that translation termination may be involved in determining whether ribosomes reinitiate at the translation start site of the same transcript for subsequent rounds of translation. In recent years, there has been a significant advancement in our knowledge of the translation termination process. The goal of this short review is to describe the state of knowledge concerning the mechanism of translation termination. Results from studies using both prokaryotic and eukaryotic systems are discussed. In particular, we attempt to address the potential role of the translation termination process in the regulation of gene expression. In addition, we discuss the modulation of the termination process as a potential target to treat diseases that arise as a consequence of mutations that prematurely terminate translation. GENERAL PRINCIPLES OF TRANSLATION TERMINATION Nearly 40 years ago, Crick and colleagues deduced the “general nature of...