Translational Read-through Inducing Drugs Research Articles

Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs).

In recent years, remarkable advances in the ability to diagnose genetic disorders have been made. The identification of disease-causing genes allows the development of gene-specific therapies with the ultimate goal to develop personalized medicines for each patient according to their own specific genetic defect. In-depth genotyping of many different genes has revealed that ~12% of inherited genetic disorders are caused by in-frame nonsense mutations. Nonsense (non-coding) mutations are caused by point mutations, which generate premature termination codons (PTCs) that cause premature translational termination of the mRNA, and subsequently inhibit normal full-length protein expression. Recently, a gene-based therapeutic approach for genetic diseases caused by nonsense mutations has emerged, namely the so-called translational read-through (TR) therapy. Read-through therapy is based on the discovery that small molecules, known as TR-inducing drugs (TRIDs), allow the translation machinery to suppress a nonsense codon, elongate the nascent peptide chain, and consequently result in the synthesis of full-length protein. Several TRIDs are currently under investigation and research has been performed on several genetic disorders caused by nonsense mutations over the years. These findings have raised hope for the usage of TR therapy as a gene-based pharmacogenetic therapy for nonsense mutations in various genes responsible for a variety of genetic diseases.

Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells.

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gβ1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients.

Translational read-through as an alternative approach for ocular gene therapy of retinal dystrophies caused by in-frame nonsense mutations.

The eye has become an excellent target for gene therapy, and gene augmentation therapy of inherited retinal disorders has made major progress in recent years. Nevertheless, a recent study indicated that gene augmentation intervention might not stop the progression of retinal degeneration in patients. In addition, for many genes, viral-mediated gene augmentation is currently not feasible due to gene size and limited packaging capacity of viral vectors as well as expression of various heterogeneous isoforms of the target gene. Thus, alternative gene-based strategies to stop or delay the retinal degeneration are necessary. This review focuses on an alternative pharmacologic treatment strategy based on the usage of translational read-through inducing drugs (TRIDs) such as PTC124, aminoglycoside antibiotics, and designer aminoglycosides for overreading in-frame nonsense mutations. This strategy has emerged as an option for up to 30-50% of all cases of recessive hereditary retinal dystrophies. In-frame nonsense mutations are single-nucleotide alterations within the gene coding sequence resulting in a premature stop codon. Consequently, translation of such mutated genes leads to the synthesis of truncated proteins, which are unable to fulfill their physiologic functions. In this context, application of TRIDs facilitates the recoding of the premature termination codon into a sense codon, thus restoring syntheses of full-length proteins. So far, clinical trials for non-ocular diseases have been initiated for diverse TRIDs. Although the clinical outcome is not analyzed in detail, an excellent safety profile, namely for PTC124, was clearly demonstrated. Moreover, recent data demonstrated sustained read-through efficacies of nonsense mutations causing retinal degeneration, as manifested in the human Usher syndrome. In addition, a strong retinal biocompatibility for PTC124 and designer aminoglycosides has been demonstrated. In conclusion, recent progress emphasizes the potential of TRIDs as an alternative pharmacologic treatment strategy for treating nonsense mutation-based retinal disorders.

Gene therapy shows potential as Usher syndrome treatment

Gene therapy shows potential as Usher syndrome treatment

Gene-based treatment options for Usher type 1C by translational read-through of a nonsense mutation

The Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. The ciliopathy is clinically and genetically heterogeneous, assigned to three clinical USH types of which the most severe type is USH1. The USH1C gene encodes the PDZ containing scaffold protein harmonin which is expressed in form of numerous alternatively spliced variants. Hamonin binds directly to all USH1/2 proteins and is a key organizer of USH protein networks in photoreceptor cells. So far no effective treatment for the ophthalmic component of USH exists. Translational read-through was introduced as an innovative therapy option for several non-ocular diseases caused by nonsense mutations leading to a premature termination stop. Here we compare the potential of translational read-through inducing drugs (TRIDs), namely PTC124 (currently in clinical phase-II for non-ocular diseases) as well as the designer aminoglycoside NB30 and NB54 as a treatment option for patients carrying a nonsense mutation in the USH1C gene causing USH1. We examined read-through in cell culture, retinal cultures and in vivo in murine retinas. Restoration of the harmonin function was tested by GST pull-downs and actin filament bundling. The TRIDs recovered functional harmonin protein and showed an excellent biocompatibility in retinal cultures with read-through vs. toxicity evidently superior for NB54 and PTC124. In vivo administration of NB54 and PTC124 to mice induced recovery of full-length harmonin. The high biocompatibility combined with the sustained read-through efficacies of these novel drugs emphasizes the potential of TRIDs in treating nonsense mutations in USH as well as in other ciliopathies.

A comparative evaluation of NB30, NB54 and PTC124 in translational read‐through efficacy for treatment of an USH1C nonsense mutation

Translational read-through-inducing drugs (TRIDs) promote read-through of nonsense mutations, placing them in the spotlight of current gene-based therapeutic research. Here, we compare for the first time the relative efficacies of new-generation aminoglycosides NB30, NB54 and the chemical compound PTC124 on retinal toxicity and read-through efficacy of a nonsense mutation in the USH1C gene, which encodes the scaffold protein harmonin. This mutation causes the human Usher syndrome, the most common form of inherited deaf-blindness. We quantify read-through efficacy of the TRIDs in cell culture and show the restoration of harmonin function. We do not observe significant differences in the read-through efficacy of the TRIDs in retinal cultures; however, we show an excellent biocompatibility in retinal cultures with read-through versus toxicity evidently superior for NB54 and PTC124. In addition, in vivo administration of NB54 and PTC124 induced recovery of the full-length harmonin a1 with the same efficacy. The high biocompatibilities combined with the sustained read-through efficacies of these drugs emphasize the potential of NB54 and PTC124 in treating nonsense mutation-based retinal disorders.