Read-through Of Nonsense Mutations Research Articles

MRNA-specific readthrough of nonsense codons by antisense oligonucleotides (R-ASOs).

Nonsense mutations account for >10% of human genetic disorders, including cystic fibrosis, Alagille syndrome, and Duchenne muscular dystrophy. A nonsense mutation results in the expression of a truncated protein, and therapeutic strategies aim to restore full-length protein expression. Most strategies under development, including small-molecule aminoglycosides, suppressor tRNAs, or the targeted degradation of termination factors, lack mRNA target selectivity and may poorly differentiate between nonsense and normal stop codons, resulting in off-target translation errors. Here, we demonstrate that antisense oligonucleotides can stimulate readthrough of disease-causing nonsense codons, resulting in high yields of full-length protein in mammalian cellular lysate. Readthrough efficiency depends on the sequence context near the stop codon and on the precise targeting position of an oligonucleotide, whose interaction with mRNA inhibits peptide release to promote readthrough. Readthrough-inducing antisense oligonucleotides (R-ASOs) enhance the potency of non-specific readthrough agents, including aminoglycoside G418 and suppressor tRNA, enabling a path toward target-specific readthrough of nonsense mutations in CFTR, JAG1, DMD, BRCA1 and other mutant genes. Finally, through systematic chemical engineering, we identify heavily modified fully functional R-ASO variants, enabling future therapeutic development.

Intravenous gentamicin therapy induces functional type VII collagen in patients with recessive dystrophic epidermolysis bullosa: an open-label clinical trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable widespread blistering skin disorder caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils. To evaluate the efficacy and safety of intravenous (IV) gentamicin readthrough therapy in patients with RDEB harbouring nonsense mutations. The primary outcomes were increased expression of C7 in patients' skin and safety assessments (ototoxicity, nephrotoxicity, autoimmune response); secondary outcomes included measuring wound healing in target wounds and assessment by a validated Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) scoring system. An open-label pilot trial to assess two different IV gentamicin regimens between August 2018 and March 2020 with follow-up through to 180 days post-treatment was carried out. Three patients with RDEB with confirmed nonsense mutations in COL7A1 in either one or two alleles and decreased baseline expression of C7 at the dermal-epidermal junction (DEJ) of their skin participated in the study. Three patients received gentamicin 7.5 mg kg-1 daily for 14 days and two of the three patients further received 7.5 mg kg-1 IV gentamicin twice weekly for 12 weeks. Patients who had pre-existing auditory or renal impairment, were currently using ototoxic or nephrotoxic medications, or had allergies to aminoglycosides or sulfate compounds were excluded. After gentamicin treatment, skin biopsies from all three patients (age range 18-28 years) exhibited increased C7 in their DEJ. With both regimens, the new C7 persisted for at least 6 months post-treatment. At 1 and 3 months post-treatment, 100% of the monitored wounds exhibited > 85% closure. Both IV gentamicin infusion regimens decreased EBDASI total activity scores. Of the patients assessed with the EBDASI, all exhibited decreased total activity scores 3 months post-treatment. All three patients completed the study; no adverse effects or anti-C7 antibodies were detected. IV gentamicin induced the readthrough of nonsense mutations in patients with RDEB and restored functional C7 in their skin, enhanced wound healing and improved clinical parameters. IV gentamicin may be a safe, efficacious, low-cost and readily available treatment for this population of patients with RDEB.

Nonsense mutation suppression is enhanced by targeting different stages of the protein synthesis process.

The introduction of premature termination codons (PTCs), as a result of splicing defects, insertions, deletions, or point mutations (also termed nonsense mutations), lead to numerous genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a large number of studies have demonstrated that certain antibiotics and other synthetic molecules can act as PTC suppressors by inducing readthrough of nonsense mutations, thereby restoring the expression of full-length proteins. Unfortunately, most PTC readthrough-inducing agents are toxic, have limited effects, and cannot be used for therapeutic purposes. Thus, further efforts are required to improve the clinical outcome of nonsense mutation suppressors. Here, by focusing on enhancing readthrough of pathogenic nonsense mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, we show that disturbing the protein translation initiation complex, as well as targeting other stages of the protein translation machinery, enhances both antibiotic and non-antibiotic-mediated readthrough of nonsense mutations. These findings strongly increase our understanding of the mechanisms involved in nonsense mutation readthrough and facilitate the development of novel therapeutic targets for nonsense suppression to restore protein expression from a large variety of disease-causing mutated transcripts.

Pharmacological induction of translational readthrough of nonsense mutations in the retinoblastoma (RB1) gene.

The retinoblastoma protein (Rb) is encoded by the RB1 tumor suppressor gene. Inactivation of RB1 by inherited or somatic mutation occurs in retinoblastoma and various other types of tumors. A significant fraction (25.9%) of somatic RB1 mutations are nonsense substitutions leading to a premature termination codon (PTC) in the RB1 coding sequence and expression of truncated inactive Rb protein. Here we show that aminoglycoside G418, a known translational readthrough inducer, can induce full-length Rb protein in SW1783 astrocytoma cells with endogenous R579X nonsense mutant RB1 as well as in MDA-MB-436 breast carcinoma cells transiently transfected with R251X, R320X, R579X or Q702X nonsense mutant RB1 cDNA. Readthrough was associated with increased RB1 mRNA levels in nonsense mutant RB1 cells. Induction of full-length Rb protein was potentiated by the cereblon E3 ligase modulator CC-90009. These results suggest that pharmacological induction of translational readthrough could be a feasible strategy for therapeutic targeting of tumors with nonsense mutant RB1.

Evaluation of Novel Enhancer Compounds in Gentamicin-Mediated Readthrough of Nonsense Mutations in Rett Syndrome.

Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients.

Balancing Nonsense Mutation Readthrough and Toxicity of Designer Aminoglycosides for Treatment of Genetic Diseases.

New derivatives of aminoglycosides with a side chain 1,2-aminoalcohol at the 5" position of ring III were designed, synthesized, and biologically evaluated. The novel lead structure (compound 6), exhibiting substantially enhanced selectivity toward eukaryotic versus prokaryotic ribosome, high readthrough activity, and considerably lower toxicity than the previous lead compounds, was discovered. Balanced readthrough activity and toxicity of 6 were demonstrated in three different nonsense DNA-constructs underlying the genetic diseases, cystic fibrosis and Usher syndrome, and in two different cell lines, baby hamster kidney and human embryonic kidney cells. Molecular dynamics simulations within the A site of the 80S yeast ribosome demonstrated a remarkable kinetic stability of 6, which potentially determines its high readthrough activity.

The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling.

Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to β-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated β-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1C R31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/β-catenin pathway.

Assessing the Therapeutic Potential of Readthrough of Nonsense Mutations in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Assessing the Therapeutic Potential of Readthrough of Nonsense Mutations in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

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

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

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

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

849 Optimization of intravenous gentamicin to restore functional laminin 332 in junctional epidermolysis bullosa patients harboring nonsense mutations

Junctional epidermolysis bullosa (JEB) is an incurable and fatal inherited blistering skin disease most commonly caused by nonsense mutations in LAMA3, LAMB3, or LAMC2 genes. These mutations impair the ability to produce functional laminin 332, needed for epidermal-dermal adherence. Previously, we demonstrated that intravenous (IV) gentamicin daily at 7.5 mg/kg for two weeks promoted readthrough of nonsense mutations and produced functional laminin 332. Here, we determined if treating the patients with higher gentamicin doses and more prolonged treatment would induce more functional laminin 332, and provide more long-term clinical improvement.

Adenoma-Derived Organoids for Precision Therapy

Human colonic organoids derived from adult tissue biopsies are based on the ability of isolated somatic epithelial stem cells to reconstitute the structure and function of the colon, offering new opportunities for studying the biology of the large intestine in both health and disease. These colonoids may also function as efficient platforms for drug screening and discovery. Here, we describe the establishment of human colonic organoids derived from healthy, and adenomatous polyp tissues. We then demonstrate that organoids grown from adenomas of familial adenomatous polyposis (FAP) patients harboring nonsense mutations in the tumor suppressor gene adenomatous polyposis coli (APC), can be used to establish a personalized therapeutic strategy which relies on nonsense mutation readthrough therapy.

Application of topical gentamicin-a new era in the treatment of genodermatosis.

The clinical use of gentamicin always lies in its antimicrobial activity in the past as an aminoglycoside antibiotic. However, in the past decade, there were considerable interests in therapeutic approaches in treating hereditary diseases. Some of the genodermatosis is caused by nonsense mutations that create premature termination codons and lead to the production of truncated or non-functional proteins. Gentamicin could induce readthrough of nonsense mutations and enable the synthesis of full-length proteins. We focus on previous publications on topical application of gentamicin and review its utility in genetic skin diseases. We search the MEDLINE through PubMed, EMBASE databases, and the Clinical Trials Registry Platform from January 1960 to July 2020 using the key search terms "gentamicin, topical gentamicin, genodermatosis, genetic skin diseases". The application of gentamicin in genodermatosis yielded promising results, both in vivo and in vitro, including Nagashima-type palmoplantar keratosis, epidermolysis bullosa, Hailey-Hailey disease, hereditary hypotrichosis simplex of the scalp, etc. CONCLUSIONS: Topical gentamicin is a potential treatment option for genodermatosis caused by nonsense mutation.

Reducing the Toxicity of Designer Aminoglycosides as Nonsense Mutation Readthrough Agents for Therapeutic Targets.

A significant proportion of genetic disease cases arise from truncation of proteins caused by premature termination codons. In eukaryotic cells some aminoglycosides cause readthrough of premature termination codons during protein translation. Inducing readthrough of these codons can potentially be of therapeutic value in the treatment of numerous genetic diseases. A significant drawback to the repeated use of aminoglycosides as treatments is the lack of balance between their readthrough efficacy and toxicity. The synthesis and biological testing of designer aminoglycoside compounds is documented herein. We disclose the implementation of a strategy to reduce cellular toxicity and maintain readthrough activity of a library of compounds by modification of the overall cationic charge of the aminoglycoside scaffold through ring I modifications.

Functional recovery of a novel knockin mouse model of dysferlinopathy by readthrough of nonsense mutation

Biallelic mutations in the dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. We found that nonsense mutations are the most common mutation type among Korean patients with dysferlinopathy; more than half of the patients have at least one nonsense allele, which may be amenable to readthrough therapy. We generated a knockin mouse, dqx, harboring DYSF p.Q832∗ mutation. Homozygous dqx mice lacked dysferlin in skeletal muscle, while 2 weeks of oral ataluren restored dysferlin expression and ameliorated skeletal muscle pathology. Their physical performance improved, and protection against eccentric contractions was noted. The improvement was most evident in mice treated with oral ataluren of 0.9 mg/mL. These improvements were sustained for 8 weeks in ataluren-treated dqx mice, while the parameters of A/J mice treated with ataluren over the same period did not improve. These results support that readthrough therapy by oral ataluren may also be applicable to dysferlinopathy patients with nonsense mutation.

ELX-02: an investigational read-through agent for the treatment of nonsense mutation-related genetic disease

ABSTRACT Introduction ELX-02, an investigational compound that is structurally an aminoglycoside analog, induces read-through of nonsense mutations through interaction with the ribosome, through which full-length functional proteins can be produced. It is being developed as a therapy for genetic diseases caused by nonsense mutations such as cystic fibrosis (CF) and nephropathic cystinosis. In Phase 1 clinical trials, 105 volunteers were exposed to ELX-02. To date, ELX-02 is well tolerated and there has been no reported treatment-related serious adverse events or deaths. Areas Covered The development of this molecule, from its pharmacology to the ongoing Phase 2 clinical trials is discussed. Expert Opinion Globally, nonsense mutations account for ~11% of all described gene lesions causing inherited monogenetic diseases. In CF and nephropathic cystinosis, they comprise from 10% to 12% of the disease-causative alleles. ELX-02 is in development as a therapeutic for patients with these alleles as in vitro and in vivo data demonstrated dose-dependent read-through of nonsense mutations to produce full-length, functional proteins. Since read-through efficiency varies between alleles and mRNA context, careful consideration of target patient populations is required. The results to date support the ongoing Phase 2 clinical evaluations of ELX-02 as a read-through agent.

2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2′-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.

Gentamicin induces COL17A1 nonsense mutation readthrough in junctional epidermolysis bullosa

Gentamicin induces <i>COL17A1</i> nonsense mutation readthrough in junctional epidermolysis bullosa

Ataluren for the Treatment of Usher Syndrome 2A Caused by Nonsense Mutations.

The identification of genetic defects that underlie inherited retinal diseases (IRDs) paves the way for the development of therapeutic strategies. Nonsense mutations caused approximately 12% of all IRD cases, resulting in a premature termination codon (PTC). Therefore, an approach that targets nonsense mutations could be a promising pharmacogenetic strategy for the treatment of IRDs. Small molecules (translational read-through inducing drugs; TRIDs) have the potential to mediate the read-through of nonsense mutations by inducing expression of the full-length protein. We provide novel data on the read-through efficacy of Ataluren on a nonsense mutation in the Usher syndrome gene USH2A that causes deaf-blindness in humans. We demonstrate Ataluren´s efficacy in both transiently USH2AG3142*-transfected HEK293T cells and patient-derived fibroblasts by restoring USH2A protein expression. Furthermore, we observed enhanced ciliogenesis in patient-derived fibroblasts after treatment with TRIDs, thereby restoring a phenotype that is similar to that found in healthy donors. In light of recent findings, we validated Ataluren´s efficacy to induce read-through on a nonsense mutation in USH2A-related IRD. In line with published data, our findings support the use of patient-derived fibroblasts as a platform for the validation of preclinical therapies. The excellent biocompatibility combined with sustained read-through efficacy makes Ataluren an ideal TRID for treating nonsense mutations based IRDs.

A no-nonsense approach to hereditary kidney disease.

The advent of a new class of aminoglycosides with increased translational readthrough of nonsense mutations and reduced toxicity offers a new therapeutic strategy for a subset of patients with hereditary kidney disease. The renal uptake and retention of aminoglycosides at a high intracellular concentration makes the kidney an ideal target for this approach. In this review, we explore the potential of aminoglycoside readthrough therapy in a number of hereditary kidney diseases and discuss the therapeutic window of opportunity for subclasses of each disease, when caused by nonsense mutations.