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.

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.

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 a-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 a-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.

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: 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.