Translational Read-through Inducing Drugs Research Articles

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression.

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman-Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named "translational readthrough" and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins' (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs.

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs

Congenital aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough-inducing drugs (TRIDs) have the ability to weaken the recognition of in-frame premature termination codons (PTCs), permitting full-length protein to be translated. We established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from two aniridia patients with prevalent PAX6 nonsense mutations. Both invitro models show reduced PAX6 protein levels, mimicking the disease. The repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP) and the positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESCs. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.

Pharmaceuticals Promoting Premature Termination Codon Readthrough: Progress in Development.

Around 11% of all known gene lesions causing human genetic diseases are nonsense mutations that introduce a premature stop codon (PTC) into the protein-coding gene sequence. Drug-induced PTC readthrough is a promising therapeutic strategy for treating hereditary diseases caused by nonsense mutations. To date, it has been found that more than 50 small-molecular compounds can promote PTC readthrough, known as translational readthrough-inducing drugs (TRIDs), and can be divided into two major categories: aminoglycosides and non-aminoglycosides. This review summarizes the pharmacodynamics and clinical application potential of the main TRIDs discovered so far, especially some newly discovered TRIDs in the past decade. The discovery of these TRIDs brings hope for treating nonsense mutations in various genetic diseases. Further research is still needed to deeply understand the mechanism of eukaryotic cell termination and drug-induced PTC readthrough so that patients can achieve the greatest benefit from the various TRID treatments.

Recoding of Nonsense Mutation as a Pharmacological Strategy.

Approximately 11% of genetic human diseases are caused by nonsense mutations that introduce a premature termination codon (PTC) into the coding sequence. The PTC results in the production of a potentially harmful shortened polypeptide and activation of a nonsense-mediated decay (NMD) pathway. The NMD pathway reduces the burden of unproductive protein synthesis by lowering the level of PTC mRNA. There is an endogenous rescue mechanism that produces a full-length protein from a PTC mRNA. Nonsense suppression therapies aim to increase readthrough, suppress NMD, or are a combination of both strategies. Therefore, treatment with translational readthrough-inducing drugs (TRIDs) and NMD inhibitors may increase the effectiveness of PTC suppression. Here we discuss the mechanism of PTC readthrough and the development of novel approaches to PTC suppression. We also discuss the toxicity and bioavailability of therapeutics used to stimulate PTC readthrough.

Influence of mRNA sequences downstream of a premature stop codon on nonsense suppression.

Premature stop codons (PSCs) arrest translation of full-length protein and trigger nonsense-mediated decay (NMD) of mRNA. Nonsense suppression by translational readthrough inducing drugs (TRIDs) like ataluren can restore protein function by inhibiting translation termination at PSCs catalyzed by release factor complex (RFC, eRF1.eRF3.GTP). We seek to understand what determines readthrough efficiency and apply our results to improve treatment of PSC diseases such as cystic fibrosis (CF). For this purpose, we utilize an in vitro translation system to examine the effects on RFC enzymatic activity of varying PSCs and downstream mRNA sequences in pretermination complexes, as measured with a high throughput fluorescence anisotropy assay and single-molecule TIRF microscopy. We have so far studied a total of seven sequences downstream from stop codons. Within this limited set, we find much lower EC50RFC values for sequences with stop codons UAG or UAA than for sequences having a UGA stop codon. These sequences display a ∼170-fold difference in measured EC50RFC values, a much larger range than the very limited range of RFC binding rates, 1.7-fold. We speculate that this difference is due, at least in part, to differences in the rate of reversible RFC dissociation. Additional sequences are currently under study. Our preliminary results explain, at least in part, why readthrough frequency is generally much higher for the UGA stop codon than for the UAG or UAA stop codons. They further suggest that combinations of drugs acting orthogonally to stimulate readthrough may be required to treat some PSC diseases, and that consideration of the mRNA sequence context flanking a nonsense codon might be critical for developing patient-specific therapeutic regimens.

Investigating the use of translational readthrough inducing drugs in keratocytes from a mouse model of brittle cornea syndrome

AbstractPurpose: Brittle Cornea Syndrome (BCS) is a rare recessive condition characterized by extreme thinning of the cornea. The collagen‐rich corneal stroma accounts for 90% of corneal thickness in humans, and is a major determinant of visual acuity. BCS results from homozygous loss‐of‐function mutations in ZNF469 (BCS1) or PRDM5 (BCS2). Our mouse model of BCS1, caused by a premature termination codon (PTC) mutation in Zfp469, showed that the stroma is thinner due to of decreased type 1 collagen expression1. To elucidate disease mechanisms and test potential therapies, we established a primary keratocyte disease‐in‐a‐dish model of BCS1. Translational readthrough inducing drugs (TRIDs) can restore full‐length proteins truncated by PTC and are a potential treatment for 20% of BCS patients with PTC mutations. Primary keratocytes from our BCS1 mouse model were treated with TRIDs to assess their efficacy in improving the BCS phenotype in vitro.Methods: Zfp469BCS1/BCS1 keratocytes were treated with TRIDs PTC124 (ataluren), amlexanox, gentamicin, geneticin or vehicle‐only, for 2 or 5 days. Following treatment, Zfp469 and Col1a1 mRNA levels were evaluated by RT‐qPCR. Changes to keratocyte cell‐derived matrices were assessed using immunostaining or the collagen dye CNA35.Results: Zfp469BCS1/BCS1 keratocytes express 40% more Zfp469 mRNA than wildtype cells; this was unchanged by treatment with amlexanox, geneticin or gentamicin. PTC124 decreased expression of Zfp469 by 50%. Expression of Col1a1, a key component of the stromal matrix, was significantly decreased by PTC124 treatment (<65% relative to vehicle‐only Zfp469BCS1/BCS1 keratocytes). Collagen deposition, visualized using CNA35 dyes, was decreased by PTC124 treatment in BCS and wildtype keratocytes.Conclusions: BCS is a devastating condition. Extreme thinning of the corneal stroma, as a result of impaired collagen production by keratocytes, makes the cornea prone to rupture. This causes irreversible sight loss. There is currently no treatment for BCS. Our initial work suggests that TRIDs may not restore functional full‐length Zfp469 in primary keratocytes and are unlikely to be of therapeutic benefit in BCS.Reference1. Stanton CM et al. A mouse model of brittle cornea syndrome caused by mutation in Zfp469. Dis Model Mech. 2021;14(9):dmm049175.

Nonsense codons suppression. An acute toxicity study of three optimized TRIDs in murine model, safety and tolerability evaluation

Stop mutations cause 11% of the genetic diseases, due to the introduction of a premature termination codon (PTC) in the mRNA, followed by the production of a truncated protein. A promising therapeutic approach is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs), restoring the expression of the protein. Recently, three new TRIDs (NV848, NV914, NV930) have been proposed, and validated by several in vitro assays, for the rescue of the CFTR protein, involved in Cystic Fibrosis disease. In this work, an acute toxicological study for the three TRIDs was conducted in vivo on mice, according to the OECD No.420 guidelines. Animals were divided into groups and treated with a single dose of TRIDs molecules or Ataluren, an FDA-approved TRID molecule, as control. Mice were observed continuously for the first day post-drugs administration and the behavioral changes were recorded. On the 15th day, animals were sacrificed for histological examinations. The results showed that acute administration of 2000 mg/kg of NV914 and Ataluren and 300 mg/kg of NV848 or NV930, did not induce any mortality within 14 days. Moreover, histopathological analysis of treated mice showed no differences when compared to the experimental controls. In summary, our results suggest a good tolerability for the three molecules, and include NV848 and NV930 in a category 4 and NV914 in a category 5 of the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals, classifying these compounds in a low-risk scale for health.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

SummaryLeber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Novel Translational Read-through-Inducing Drugs as a Therapeutic Option for Shwachman-Diamond Syndrome.

Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. Besides confirming that ataluren can efficiently induce SBDS protein re-expression in SDS cells, the authors found that another analogue, namely NV848, can restore full-length SBDS protein synthesis as well, showing very low toxicity in zebrafish. Furthermore, NV848 can improve myeloid differentiation in bone marrow hematopoietic progenitors, enhancing neutrophil maturation and reducing the number of dysplastic granulocytes in vitro. Therefore, these findings broaden the possibilities of developing novel therapeutic options in terms of nonsense mutation suppression for SDS. Eventually, this study may act as a proof of concept for the development of similar approaches for other IBMFS caused by nonsense mutations.

Translational readthrough of ciliopathy genes BBS2 and ALMS1 restores protein, ciliogenesis and function in patient fibroblasts.

BackgroundCiliary dysfunction underlies a range of genetic disorders collectively termed ciliopathies, for which there are no treatments available. Bardet-Biedl syndrome (BBS) is characterised by multisystemic involvement, including rod-cone dystrophy and renal abnormalities. Together with Alström syndrome (AS), they are known as the ‘obesity ciliopathies’ due to their common phenotype. Nonsense mutations are responsible for approximately 11% and 40% of BBS and AS cases, respectively. Translational readthrough inducing drugs (TRIDs) can restore full-length protein bypassing in-frame premature termination codons, and are a potential therapeutic approach for nonsense-mediated ciliopathies.MethodsPatient fibroblasts harbouring nonsense mutations from two different ciliopathies (Bardet-Biedl Syndrome and Alström Syndrome) were treated with PTC124 (ataluren) or amlexanox. Following treatment, gene expression, protein levels and ciliogenesis were evaluated. The expression of intraflagellar transport protein IFT88 and G-protein coupled receptor SSTR3 was investigated as a readout of ciliary function.FindingsmRNA expression was significantly increased in amlexanox-treated patient fibroblasts, and full-length BBS2 or ALMS1 protein expression was restored in PTC124- and amlexanox-treated fibroblasts. Treatment with TRIDs significantly improved ciliogenesis defects in BBS2Y24*/R275* fibroblasts. Treatment recovered IFT88 expression and corrected SSTR3 mislocalisation in BBS2Y24*/R275* and ALMS1S1645*/S1645* fibroblasts, suggesting rescue of ciliary function.InterpretationThe recovery of full-length BBS2 and ALMS1 expression and correction of anatomical and functional ciliary defects in BBS2Y24*/R275* and ALMS1S1645*/S1645* fibroblasts suggest TRIDs are a potential therapeutic option for the treatment of nonsense-mediated ciliopathies.

Stop Codon Context-Specific Induction of Translational Readthrough.

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC)—the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems.

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after treatment with the selected molecules in cells expressing nonsense–CFTR–mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

Pharmacophore-Based Design of New Chemical Scaffolds as Translational Readthrough-Inducing Drugs (TRIDs).

Translational readthrough-inducing drugs (TRIDs) rescue the functional full-length protein expression in genetic diseases, such as cystic fibrosis, caused by premature termination codons (PTCs). Small molecules have been developed as TRIDs to trick the ribosomal machinery during recognition of the PTC. Herein we report a computational study to identify new TRID scaffolds. A pharmacophore approach was carried out on compounds that showed readthrough activity. The pharmacophore model applied to screen different libraries containing more than 87000 compounds identified four hit-compounds presenting scaffolds with diversity from the oxadiazole lead. These compounds have been synthesized and tested using the Fluc reporter harboring the UGA PTC. Moreover, the cytotoxic effect and the expression of the CFTR protein were evaluated. These compounds, a benzimidazole derivative (NV2899), a benzoxazole derivative (NV2913), a thiazole derivative (NV2909), and a benzene-1,3-disulfonate derivative (NV2907), were shown to be potential new lead compounds as TRIDs, boosting further efforts to address the optimization of the chemical scaffolds.

Strategies against Nonsense: Oxadiazoles as Translational Readthrough-Inducing Drugs (TRIDs).

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs.

Rescuing the CFTR protein function: Introducing 1,3,4-oxadiazoles as translational readthrough inducing drugs

Nonsense mutations in the CFTR gene prematurely terminate translation of the CFTR mRNA leading to the production of a truncated protein that lacks normal function causing a more severe form of the cystic fibrosis (CF) disease. About 10% of patients affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by Translational Readthrough Inducing Drugs (TRIDs) such as PTC124. In this context we aimed to compare the activity of PTC124 with analogues differing in the heteroatoms position in the central heterocyclic core. By a validated protocol consisting of computational screening, synthesis and biological tests we identified a new small molecule (NV2445) with 1,3,4-oxadiazole core showing a high readthrough activity. Moreover, we evaluated the CFTR functionality after NV2445 treatment in CF model systems and in cells expressing a nonsense-CFTR-mRNA. Finally, we studied the supramolecular interactions between TRIDs and CFTR-mRNA to assess the biological target/mechanism and compared the predicted ADME properties of NV2445 and PTC124.

The effect of PTC124 on choroideremia fibroblasts and iPSC-derived RPE raises considerations for therapy

Inherited retinal dystrophies (IRDs) are caused by mutations in over 200 genes, resulting in a range of therapeutic options. Translational read-through inducing drugs (TRIDs) offer the possibility of treating multiple IRDs regardless of the causative gene. TRIDs promote ribosomal misreading of premature stop codons, which results in the incorporation of a near-cognate amino acid to produce a full-length protein. The IRD choroideremia (CHM) is a pertinent candidate for TRID therapy, as nonsense variants cause 30% of cases. Recently, treatment of the UAA nonsense-carrying CHM zebrafish model with the TRID PTC124 corrected the underlying biochemical defect and improved retinal phenotype. To be clinically relevant, we studied PTC124 efficiency in UAA nonsense-carrying human fibroblasts and induced pluripotent stem cell-derived retinal pigment epithelium, as well as in a UAA-mutated CHM overexpression system. We showed that PTC124 treatment induces a non-significant trend for functional rescue, which could not be improved by nonsense-mediated decay inhibition. Furthermore, it does not produce a detectable CHM-encoded protein even when coupled with a proteasome inhibitor. We suggest that drug efficiency may depend upon on the target amino acid and its evolutionary conservation, and argue that patient cells should be screened in vitro prior to inclusion in a clinical trial.

The Usher Syndrome, a Human Ciliopathy

The human Usher syndrome (USH) is a complex, rare disease manifesting in its most common form of inherited deaf-blindness. Due to the heterogeneous manifestation of the clinical symptoms, three clinical types (USH1-3) are distinguished according to the severity of the disease pattern. For a correct diagnosis, in addition to the auditory tests in early newborn screening, ophthalmological examinations and molecular genetic analysis are important. Ten known USH genes encode proteins, which are from heterogeneous protein families, interact in functional protein networks. In the eye and in the ear, USH proteins are expressed primarily in the mechano-sensitive hair cells and the rod and cone photoreceptor cells, respectively. In the hair cells, the USH protein networks are essential for the correct differentiation of the hair bundles as well as for the function of the mechano-electrical transduction complex in the matured cell. In the photoreceptor cells, USH proteins are located in the ciliary region and participate in intracellular transport processes. In addition, a USH protein network is present in the so-called calyceal processes. The lack of calyceal processes and the absence of a prominent visual phenotype in the mouse disqualifies mice as models for studies on the ophthalmic component of USH. While hearing impairments can be compensated with hearing aids and cochlear implants, there is no practical therapy for USH in the eye. Currently, gene-based therapy concepts, such as gene addition, applications of antisense oligonucleotides and TRIDs ("translational readthrough inducing drugs") for the readthrough of nonsense mutations are preclinically evaluated. For USH1B/MYO7A the UshStat gene therapy clinical trial is ongoing.

Arl3 and RP2 regulate the trafficking of ciliary tip kinesins.

Ciliary trafficking defects are the underlying cause of many ciliopathies, including Retinitis Pigmentosa (RP). Anterograde intraflagellar transport (IFT) is mediated by kinesin motor proteins; however, the function of the homodimeric Kif17 motor in cilia is poorly understood, whereas Kif7 is known to play an important role in stabilizing cilia tips. Here we identified the ciliary tip kinesins Kif7 and Kif17 as novel interaction partners of the small GTPase Arl3 and its regulatory GTPase activating protein (GAP) Retinitis Pigmentosa 2 (RP2). We show that Arl3 and RP2 mediate the localization of GFP-Kif17 to the cilia tip and competitive binding of RP2 and Arl3 with Kif17 complexes. RP2 and Arl3 also interact with another ciliary tip kinesin, Kif7, which is a conserved regulator of Hedgehog (Hh) signaling. siRNA-mediated loss of RP2 or Arl3 reduced the level of Kif7 at the cilia tip. This was further validated by reduced levels of Kif7 at cilia tips detected in fibroblasts and induced pluripotent stem cell (iPSC) 3D optic cups derived from a patient carrying an RP2 nonsense mutation c.519C > T (p.R120X), which lack detectable RP2 protein. Translational read-through inducing drugs (TRIDs), such as PTC124, were able to restore Kif7 levels at the ciliary tip of RP2 null cells. Collectively, our findings suggest that RP2 and Arl3 regulate the trafficking of specific kinesins to cilia tips and provide additional evidence that TRIDs could be clinically beneficial for patients with this retinal degeneration.