Extended stop codon context predicts nonsense codon readthrough efficiency in human cells.

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.

Gene-targeted therapy for neurofibromatosis and schwannomatosis: The path to clinical trials.

Numerous successful gene-targeted therapies are arising for the treatment of a variety of rare diseases. At the same time, current treatment options for neurofibromatosis 1 and schwannomatosis are limited and do not directly address loss of gene/protein function. In addition, treatments have mostly focused on symptomatic tumors, but have failed to address multisystem involvement in these conditions. Gene-targeted therapies hold promise to address these limitations. However, despite intense interest over decades, multiple preclinical and clinical issues need to be resolved before they become a reality. The optimal approaches to gene-, mRNA-, or protein restoration and to delivery to the appropriate cell types remain elusive. Preclinical models that recapitulate manifestations of neurofibromatosis 1 and schwannomatosis need to be refined. The development of validated assays for measuring neurofibromin and merlin activity in animal and human tissues will be critical for early-stage trials, as will the selection of appropriate patients, based on their individual genotypes and risk/benefit balance. Once the safety of gene-targeted therapy for symptomatic tumors has been established, the possibility of addressing a wide range of symptoms, including non-tumor manifestations, should be explored. As preclinical efforts are underway, it will be essential to educate both clinicians and those affected by neurofibromatosis 1/schwannomatosis about the risks and benefits of gene-targeted therapy for these conditions.

RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay.

Background: CFTR nonsense alleles generate negligible CFTR protein due to the nonsense mutation: 1) triggering CFTR mRNA degradation by nonsense-mediated mRNA decay (NMD), and 2) terminating CFTR mRNA translation prematurely. Thus, people with cystic fibrosis (PwCF) who carry nonsense alleles cannot benefit from current modulator drugs, which target CFTR protein. In this study, we examined whether PTBP1 and HNRNPL, two RNA binding proteins that protect a subset of mRNAs with a long 3' untranslated region (UTR) from NMD, similarly affect CFTR mRNA.Silencing RNAs were used to deplete PTBP1 or HNRNPL in 16HBE14o- human bronchial epithelial cells expressing WT, G542X, or W1282X CFTR. CFTR mRNA abundance was measured relative to controls by quantitative PCR. PTBP1 and HNRNPL were also exogenously expressed in each cell line and CFTR mRNA levels were similarly quantified. Results: PTBP1 depletion reduced CFTR mRNA abundance in all three 16HBE14o- cell lines; HRNPL depletion reduced CFTR mRNA abundance in only the G542X and W1282X cell lines. Notably, decreased CFTR mRNA abundance correlated with increased mRNA decay. Exogenous expression of PTBP1 or HNRNPL increased CFTR mRNA abundance in all three cell lines; HNRNPL overexpression generally increased CFTR to a greater extent in G542X and W1282X 16HBE14o- cells.Our data indicate that PTBP1 and HNRNPL regulate CFTR mRNA abundance by protecting CFTR transcripts from NMD. This suggests that PTBP1 and/or HNRNPL may represent potential therapeutic targets to increase CFTR mRNA abundance and enhance responses to CFTR modulators and other therapeutic approaches in PwCF.

Discovery of dysregulated circular RNAs in whole blood transcriptomes from cystic fibrosis patients - implication of a role for cellular senescence in cystic fibrosis.

BACKGROUND: A largely unexplored area of research is the identification and characterization of circular RNA (circRNA) in cystic fibrosis (CF). This study is the first to identify and characterize alterations in circRNA expression in cells lacking CFTR function. The circRNA expression profiles in whole blood transcriptomes from CF patients homozygous for the pathogenetic variant F508delCFTR are compared to healthy controls. METHODS: We developed a circRNA pipeline called circRNAFlow utilizing Nextflow. Whole blood transcriptomes from CF patients homozygous for the F508delCFTR-variant and healthy controls were utilized as input to circRNAFlow to discover dysregulated circRNA expression in CF samples compared to wild-type controls. Pathway enrichment analyzes were performed to investigate potential functions of dysregulated circRNAs in whole blood transcriptomes from CF samples compared to wild-type controls. RESULTS: A total of 118 dysregulated circRNAs were discovered in whole blood transcriptomes from CF patients homozygous for the F508delCFTR variant compared to healthy controls. 33 circRNAs were up regulated whilst 85 circRNAs were down regulated in CF samples compared to healthy controls. The overrepresented pathways of the host genes harboring dysregulated circRNA in CF samples compared to controls include positive regulation of responses to endoplasmic reticulum stress, intracellular transport, protein serine/threonine kinase activity, phospholipid-translocating ATPase complex, ferroptosis and cellular senescence. These enriched pathways corroborate the role of dysregulated cellular senescence in CF. CONCLUSION: This study highlights the underexplored roles of circRNAs in CF with a perspective to provide a more complete molecular characterization of CF.

The synthetic aminoglycoside ELX-02 induces readthrough of G550X-CFTR producing superfunctional protein that can be further enhanced by CFTR modulators.

Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl- conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy.NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.

Triamterene Functions as an Effective Nonsense Suppression Agent for MPS I-H (Hurler Syndrome).

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.

Ataluren suppresses a premature termination codon in an MPS I-H mouse.

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.

A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion.

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.

A regulated NMD mouse model supports NMD inhibition as a viable therapeutic option to treat genetic diseases.

Nonsense-mediated mRNA decay (NMD) targets mRNAs that contain a premature termination codon (PTC) for degradation, preventing their translation. By altering the expression of PTC-containing mRNAs, NMD modulates the inheritance pattern and severity of genetic diseases. NMD also limits the efficiency of suppressing translation termination at PTCs, an emerging therapeutic approach to treat genetic diseases caused by in-frame PTCs (nonsense mutations). Inhibiting NMD may help rescue partial levels of protein expression. However, it is unclear whether long-term, global NMD attenuation is safe. We hypothesize that a degree of NMD inhibition can be safely tolerated after completion of prenatal development. To test this hypothesis, we generated a novel transgenic mouse that expresses an inducible, dominant-negative form of human UPF1 (dnUPF1) to inhibit NMD in mouse tissues by different degrees, allowing us to examine the effects of global NMD inhibition in vivo A thorough characterization of these mice indicated that expressing dnUPF1 at levels that promote relatively moderate to strong NMD inhibition in most tissues for a 1-month period produced modest immunological and bone alterations. In contrast, 1 month of dnUPF1 expression to promote more modest NMD inhibition in most tissues did not produce any discernable defects, indicating that moderate global NMD attenuation is generally well tolerated in non-neurological somatic tissues. Importantly, a modest level of NMD inhibition that produced no overt abnormalities was able to significantly enhance in vivo PTC suppression. These results suggest that safe levels of NMD attenuation are likely achievable, and this can help rescue protein deficiencies resulting from PTCs.

Pharmacological approaches for targeting cystic fibrosis nonsense mutations.

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder. The clinical manifestations of the disease are caused by ∼2,000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. It is unlikely that any one approach will be efficient in correcting all defects. The recent approvals of ivacaftor, lumacaftor/ivacaftor and elexacaftor/tezacaftor/ivacaftor represent the genesis of a new era of precision combination medicine for the CF patient population. In this review, we discuss targeted translational readthrough approaches as mono and combination therapies for CFTR nonsense mutations. We examine the current status of efficacy of translational readthrough/nonsense suppression therapies and their limitations, including non-native amino acid incorporation at PTCs and nonsense-mediated mRNA decay (NMD), along with approaches to tackle these limitations. We further elaborate on combining various therapies such as readthrough agents, NMD inhibitors, and corrector/potentiators to improve the efficacy and safety of suppression therapy. These mutation specific strategies that are directed towards the basic CF defects should positively impact CF patients bearing nonsense mutations.

Mutation-Directed Therapeutics for Neurofibromatosis Type I.

Significant advances in biotechnology have led to the development of a number of different mutation-directed therapies. Some of these techniques have matured to a level that has allowed testing in clinical trials, but few have made it to approval by drug-regulatory bodies for the treatment of specific diseases. While there are still various hurdles to be overcome, recent success stories have proven the potential power of mutation-directed therapies and have fueled the hope of finding therapeutics for other genetic disorders. In this review, we summarize the state-of-the-art of various therapeutic approaches and assess their applicability to the genetic disorder neurofibromatosis type I (NF1). NF1 is caused by the loss of function of neurofibromin, a tumor suppressor and downregulator of the Ras signaling pathway. The condition is characterized by a variety of phenotypes and includes symptoms such as skin spots, nervous system tumors, skeletal dysplasia, and others. Hence, depending on the patient, therapeutics may need to target different tissues and cell types. While we also discuss the delivery of therapeutics, in particular via viral vectors and nanoparticles, our main focus is on therapeutic techniques that reconstitute functional neurofibromin, most notably cDNA replacement, CRISPR-based DNA repair, RNA repair, antisense oligonucleotide therapeutics including exon skipping, and nonsense suppression.

Finding sense in the context.

Ribosomal profiling has shed new light on how ribosomes can ignore stop codons in messenger RNA.

Identification of the amino acids inserted during suppression of CFTR nonsense mutations and determination of their functional consequences.

In-frame premature termination codons (PTCs) account for ∼11% of all disease-associated mutations. PTC suppression therapy utilizes small molecules that suppress translation termination at a PTC to restore synthesis of a full-length protein. PTC suppression is mediated by the base pairing of a near-cognate aminoacyl-tRNA with a PTC and subsequently, the amino acid becomes incorporated into the nascent polypeptide at the site of the PTC. However, little is known about the identity of the amino acid(s) inserted at a PTC during this process in mammalian cells, or how the surrounding sequence context influences amino acid incorporation. Here, we determined the amino acids inserted at the cystic fibrosis transmembrane conductance regulator (CFTR) W1282X PTC (a UGA codon) in the context of its three upstream and downstream CFTR codons during G418-mediated suppression. We found that leucine, cysteine and tryptophan are inserted during W1282X suppression. Interestingly, these amino acids (and their proportions) are significantly different from those recently identified following G418-mediated suppression of the CFTR G542X UGA mutation. These results demonstrate for the first time that local mRNA sequence context plays a key role in near-cognate aminoacyl-tRNA selection during PTC suppression. We also found that some variant CFTR proteins generated by PTC suppression exhibit reduced maturation and activity, indicating the complexity of nonsense suppression therapy. However, both a CFTR corrector and potentiator enhanced activity of protein variants generated by G418-mediated suppression. These results suggest that PTC suppression in combination with CFTR modulators may be beneficial for the treatment of CF patients with PTCs.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote 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.

Discovery of Clinically Approved Agents That Promote Suppression of Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations.

RATIONALE: Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic. OBJECTIVES: To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs. METHODS: Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents. MEASUREMENTS AND MAIN RESULTS: From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro. CONCLUSIONS: Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.

Nonsense Suppression as an Approach to Treat Lysosomal Storage Diseases.

In-frame premature termination codons (PTCs) (also referred to as nonsense mutations) comprise ~10% of all disease-associated gene lesions. PTCs reduce gene expression in two ways. First, PTCs prematurely terminate translation of an mRNA, leading to the production of a truncated polypeptide that often lacks normal function and/or is unstable. Second, PTCs trigger degradation of an mRNA by activating nonsense-mediated mRNA decay (NMD), a cellular pathway that recognizes and degrades mRNAs containing a PTC. Thus, translation termination and NMD are putative therapeutic targets for the development of treatments for genetic diseases caused by PTCs. Over the past decade, significant progress has been made in the identification of compounds with the ability to suppress translation termination of PTCs (also referred to as readthrough). More recently, NMD inhibitors have also been explored as a way to enhance the efficiency of PTC suppression. Due to their relatively low threshold for correction, lysosomal storage diseases are a particularly relevant group of diseases to investigate the feasibility of nonsense suppression as a therapeutic approach. In this review, the current status of PTC suppression and NMD inhibition as potential treatments for lysosomal storage diseases will be discussed.

Therapeutics based on stop codon readthrough.

Nonsense suppression therapy encompasses approaches aimed at suppressing translation termination at in-frame premature termination codons (PTCs, also known as nonsense mutations) to restore deficient protein function. In this review, we examine the current status of PTC suppression as a therapy for genetic diseases caused by nonsense mutations. We discuss what is currently known about the mechanism of PTC suppression as well as therapeutic approaches under development to suppress PTCs. The approaches considered include readthrough drugs, suppressor tRNAs, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay. We also discuss the barriers that currently limit the clinical application of nonsense suppression therapy and suggest how some of these difficulties may be overcome. Finally, we consider how PTC suppression may play a role in the clinical treatment of genetic diseases caused by nonsense mutations.

Long-term nonsense suppression therapy moderates MPS I-H disease progression.

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 Idua(tm1Kmke) 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 Idua(tm1Kmke) mice throughout a 28-week treatment period. An examination of more complex MPS I-H phenotypes in Idua(tm1Kmke) 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.

Attenuation of nonsense-mediated mRNA decay enhances in vivo nonsense suppression.

Nonsense suppression therapy is an approach to treat genetic diseases caused by nonsense mutations. This therapeutic strategy pharmacologically suppresses translation termination at Premature Termination Codons (PTCs) in order to restore expression of functional protein. However, the process of Nonsense-Mediated mRNA Decay (NMD), which reduces the abundance of mRNAs containing PTCs, frequently limits this approach. Here, we used a mouse model of the lysosomal storage disease mucopolysaccharidosis I-Hurler (MPS I-H) that carries a PTC in the Idua locus to test whether NMD attenuation can enhance PTC suppression in vivo. Idua encodes alpha-L-iduronidase, an enzyme required for degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. We found that the NMD attenuator NMDI-1 increased the abundance of the PTC-containing Idua transcript. Furthermore, co-administration of NMDI-1 with the PTC suppression drug gentamicin enhanced alpha-L-iduronidase activity compared to gentamicin alone, leading to a greater reduction of GAG storage in mouse tissues, including the brain. These results demonstrate that NMD attenuation significantly enhances suppression therapy in vivo.

Suppression of premature termination codons as a therapeutic approach.

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.