Nonsense suppression rescues functional protein from mRNAs containing in-frame premature termination codons (PTCs). This approach employs small molecules that promote insertion of tRNAs at a PTC during translation (called "read-through"), enabling translation to continue past the PTC to generate a full-length, functional protein. Some aminoglycosides such as G418 promote readthrough; however, their long-term use is thwarted by toxicity and modest potency. ELX-02, a novel synthetic aminoglycoside, was developed to have both enhanced readthrough activity and reduced toxicity in mammalian cells compared to traditional aminoglycosides. Recent studies suggest that at safe doses, aminoglycosides like ELX-02 may not possess sufficient efficacy to provide clinical benefits for most diseases. "Enhancer" molecules have been identified that do not possess readthrough activity themselves but enhance the efficiency of aminoglycoside-mediated read-through. In this study, three newly identified series of enhancer molecules were tested for the ability to stimulate aminoglycoside-mediated readthrough of PTCs associated with cystic fibrosis and neurofibromatosis type 1. Although none of the enhancers alone induced readthrough, they all significantly increased readthrough via aminoglycosides at low doses (EC10). Overall, our results suggest that enhancer compounds may be a viable way to overcome potency issues associated with aminoglycosides, enabling rescue of protein function from nonsense alleles while minimizing toxicity.

Protein synthesis terminates when a stop codon enters the ribosome’s A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3’-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient’s likelihood of improvement in response to drugs given their nonsense mutation sequence context.

Neurofibromatosis type 1 (NF1) results from germline mutations in the tumor-suppressor gene NF1 and predisposes patients to developing nervous system tumors. Twenty percent of NF1 patients harbor nonsense mutations resulting in premapies can facilitate ribosomal readthrough of PTCs to restore full-length protein, but their potential in NF1 is underexplored. We developed a minipig model of NF1 carrying a PTC to test whether nonsense suppression could restore expression of the NF1-encoded protein neurofibromin in vitro and in vivo. Nonsense suppression did not reliably increase neurofibromin in primary NF1-'- Schwann cells isolated from minipig neurofibromas but could reduce phosphorylated ERK. Gentamicin in vivo produced a similar plasma pharmacokinetic profile to humans and was detectable in clinically relevant tissues, including cerebral cortex, sciatic nerve, optic nerve, and skin. In gentamicin-treated animals, increased neurofibromin expression was seen in the optic of transcripts with PTCs, which could impede nonsense neurofibromas. Thus, the effectiveness of nonsense suppresof NMD inhibitors.

Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene's diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC.

Abstarct Suppressing translation termination at premature termination codons (PTCs), termed readthrough, is a potential therapy for genetic diseases caused by nonsense mutations. Ataluren is a compound that has shown promise for clinical use as a readthrough agent. However, some reports suggest that ataluren is ineffective at suppressing PTCs. To further evaluate the effectiveness of ataluren as a readthrough agent, we examined its ability to suppress PTCs in a variety of previously untested models. Using NanoLuc readthrough reporters expressed in two different cell types, we found that ataluren stimulated a significant level of readthrough. We also explored the ability of ataluren to suppress a nonsense mutation associated with Mucopolysaccharidosis I-Hurler (MPS I-H), a genetic disease that is caused by a deficiency of α-L-iduronidase that leads to lysosomal accumulation of glycosaminoglycans (GAGs). Using mouse embryonic fibroblasts (MEFs) derived from Idua-W402X mice, we found that ataluren partially rescued α-L-iduronidase function and significantly reduced GAG accumulation relative to controls. Two-week oral administration of ataluren to Idua-W402X mice led to significant GAG reductions in most tissues compared to controls. Together, these data reveal important details concerning the efficiency of ataluren as a readthrough agent and the mechanisms that govern its ability to suppress PTCs. Key messages Ataluren promotes readthrough of PTCs in a wide variety of contexts. Ataluren reduces glycosaminoglyan storage in MPS I-H cell and mouse models. Ataluren has a bell-shaped dose–response curve and a narrow effective range.

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

Bacterial protein synthesis initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. We have demonstrated the application of this system to obtain N-formylated form of a mycobacterial secretory protein ESAT-6 which is an important target for T-cell recognition during Mycobacterium tuberculosis infection. The gene encoding the ESAT-6 of M. tuberculosis was inserted into a bacterial expression vector pET23a (+) resulting in a 6 × His-esat-6 fusion gene construction with histidine tag at its C-terminus. This recombinant plasmid was transformed into Escherichia coli strain BL21(DE3) and effectively expressed in presence or absence of a peptide deformylase inhibitor, actinonin. The expressed fusion proteins (non-formylated and N-formylated ESAT-6) found in the inclusion bodies were purified by Ni2+–NTA chromatography. The N-terminal formylation of ESAT-6 was confirmed by advanced proteomic techniques including MALDI-TOF mass spectrometry and 2-DE. The gene encoding ESAT-6 of M. tuberculosis was successfully cloned and expressed in E. coli. The N-terminal formylation of this protein (ESAT-6) was carried out by inducing the recombinant clone harboring the ESAT-6 gene in presence of actinonin (peptide deformylase inhibitor).

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/-) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR-/-) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR-/- rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR-/- males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR-/- animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities.

Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame premature termination codons (PTCs; also commonly known as nonsense mutations). This approach utilizes compounds that suppress translation termination at PTCs, which allows translation to continue and partial levels of deficient protein function to be restored. We hypothesize that suppression therapy can attenuate the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H), the severe form of α-l-iduronidase deficiency. α-l-iduronidase participates in glycosaminoglycan (GAG) catabolism and its insufficiency causes progressive GAG accumulation and onset of the MPS I-H phenotype, which consists of multiple somatic and neurological defects. 60–80% of MPS I-H patients carry a nonsense mutation in the IDUA gene. We previously showed that 2-week treatment with the designer aminoglycoside NB84 restored enough α-l-iduronidase function via PTC suppression to reduce tissue GAG accumulation in the Iduatm1Kmke MPS I-H mouse model, which carries a PTC homologous to the human IDUA-W402X nonsense mutation. Here we report that long-term NB84 administration maintains α-l-iduronidase activity and GAG reduction in Iduatm1Kmke mice throughout a 28-week treatment period. An examination of more complex MPS I-H phenotypes in Iduatm1Kmke mice following 28-week NB84 treatment revealed significant moderation of the disease in multiple tissues, including the brain, heart and bone, that are resistant to current MPS I-H therapies. This study represents the first demonstration that long-term nonsense suppression therapy can moderate progression of a genetic disease.

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

We are exploring the use of pharmacological agents to suppress nonsense mutations that cause genetic diseases. This approach could theoretically restore the synthesis of full length, functional proteins and relieve disease phenotypes. Much of our work focuses on cystic fibrosis (CF), a life‐threatening genetic disease that compromises function of the lungs and pancreas. CF is caused by mutations in the CFTR gene, which encodes the CFTR protein, a cAMP‐activated chloride channel. In cultured cells, we have shown that readthrough agents such as gentamicin and ataluren (formerly PTC124®) suppress CFTR nonsense mutations, resulting in a partial restoration of CFTR protein and function. Using a transgenic mouse model expressing the human CFTR gene with the G542X nonsense mutation found in CF patients, we have shown that these compounds can restore human CFTR expression and function in vivo. Recent data from a phase II clinical study support these conclusions, and show that ataluren can reduce the electrophysiological defects associated with CFTR nonsense mutations in CF patients. This therapeutic approach is also currently being applied to Duchenne Muscular Dystrophy and the lysosomal storage disease Hurler syndrome (MPS I‐H). Together, these results suggest that this approach could benefit a genotypic subset of patients with a wide range of genetic diseases caused by nonsense mutations. Supported by NIH RO1 NS057412 and P30 DK072482.

Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-L-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20-40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr(-/-) hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach.

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.

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.

Alarge number of diseases are caused by premature stop mutations that often lead to a complete loss of protein function and a severe reduction in mRNA levels due to nonsense-mediated mRNA decay (NMD). Two main approaches to develop treatments for diseases caused by premature stop mutations have been investigated. The first is to reduce the efficiency of translation termination through the use of pharmacological agents or by the expression of suppressor tRNAs. The second approach is to replace the premature stop mutation with wild-type sequence using gene repair techniques. Although each of these approaches have been demonstrated using in vitro studies or mouse models, currently only strategies using pharmacological agents to suppress stop mutations have reached preliminary clinical trials. The future of suppression therapy will require finding ways to increase the efficacy of current compounds to suppress premature stop mutations without side effects, or to design or discover safe new compounds that suppress premature stop mutations with increased efficiency. In addition, combined therapies that simultaneously suppress a premature stop mutation and inhibit NMD of the nonsense-containing mRNA may be the most effective way to increase the efficiency of suppression therapy.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease cystic fibrosis. We previously reported that gentamicin administration suppressed a CFTR premature stop mutation in a Cftr−/− mouse model carrying a human CFTR-G542X (hCFTR-G542X) transgene, resulting in the appearance of hCFTR protein and function. However, the high doses used in that study resulted in peak serum levels well beyond the levels typically administered to humans. To address this problem, we identified doses of both gentamicin and amikacin that resulted in peak serum levels within their accepted clinical ranges. We then asked whether these doses could suppress the hCFTR-G542X mutation in the Cftr−/− hCFTR-G542X mouse model. Our results indicate that low doses of each compound restored some hCFTR protein expression and function, as shown by immunofluorescence and short-circuit current measurements. However, we found that amikacin suppressed the hCFTR-G542X premature stop mutation more effectively than gentamicin when administered at these clinically relevant doses. Because amikacin is also less toxic than gentamicin, it may represent a superior choice for suppression therapy in patients that carry a premature stop mutation in the CFTR gene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since ~5% of all mutant CF alleles are stop mutations, it can be calculated that ~10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr–/–) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr–/–hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.