PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model

Abstract

Nonsense mutations inactivate gene function and are the underlying cause of a large percentage of the individual cases of many genetic disorders. PTC124 is an orally bioavailable compound that promotes readthrough of premature translation termination codons, suggesting that it may have the potential to treat genetic diseases caused by nonsense mutations. Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr-/- mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Translational readthrough of the premature stop codon was demonstrated in this mouse model in two ways. First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr-/- hCFTR-G542X mice. In addition, functional assays demonstrated that PTC124 treatment restored 24-29% of the average cAMP-stimulated transepithelial chloride currents observed in wild-type mice. These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. In light of its oral bioavailability, safety toxicology profile in animal studies, and efficacy with other nonsense alleles, PTC124 has the potential to be an important therapeutic agent for the treatment of inherited diseases caused by nonsense mutations.

Fig. 1.

hCFTR expression in submucosal glands of intestinal tissues from mice treated with PTC124 by once daily s.c. injections. Samples were prepared from the duodenum of untreated Cftr−/− hCFTR-G542X mice and Cftr−/− hCFTR-G542X mice treated with 15, 30, or 60 mg/kg PTC124. Intestinal tissues from Cftr−/− hCFTR-G542X mice treated with 34 mg/kg gentamicin by the same administration protocol were also examined as a positive control. For immunofluorescence assays, samples were incubated with either preimmune or hCFTR-NBD1 serum. After incubation of the sample with a fluorescent secondary antibody, the samples were visualized by fluorescence microscopy. (Magnification, ×400.)

Fig. 2.

Effect of PTC124 s.c. injection on cAMP-stimulated transepithelial chloride currents in intestinal tissues from Cftr−/− hCFTR-G542X and Cftr+/+ mice. (Upper) Individual data points from short-circuit current measurements. (Lower) Analysis of the data. P values comparing all data points from treated and untreated mice were calculated with Student's t test. P < 0.05 was considered significant.

Fig. 3.

Comparison of serum levels of PTC124 after s.c. vs. oral administration. (A) Blood was taken from different age-matched Cftr+/− hCFTR-G542X mice at the indicated times after s.c. injection of 60 mg/kg PTC124. (B) Blood samples were taken after feeding Cftr+/− hCFTR-G542X mice a liquid diet containing 0.3 or 0.9 mg/ml PTC124 for 2–6 days or Cftr−/− hCFTR-G542X mice a liquid diet containing 0.3 or 0.9 mg/ml PTC124 for 14–21 days. The concentration of PTC124 in each serum sample was determined by liquid chromatography/tandem mass spectrometry (for details, see Materials and Methods).

Fig. 4.

Immunofluorescence of hCFTR in the submucosal glands of intestinal tissues from mice fed a liquid diet containing PTC124. Samples were from the duodenum of Cftr−/− hCFTR-G542X mice treated with 0.3 or 0.9 mg/ml PTC124 in the Peptamen liquid diet. Samples were incubated with either preimmune or hCFTR-NBD1 serum as indicated. After incubation of the sample with a fluorescent secondary antibody, the samples were visualized by fluorescence microscopy.

Fig. 5.

Effect of PTC124 oral administration on cAMP-stimulated transepithelial chloride currents in intestinal tissues from Cftr−/− hCFTR-G542X, Cftr+/+, and Cftr−/− mice. (Upper) Individual data points from short-circuit current measurements. (Lower) Analysis of the data. P values comparing all data points from treated and untreated mice were calculated with Student's t test. P < 0.05 was considered significant.

Fig. 6.

RT-PCR detection of hCFTR mRNA in untreated Cftr−/− hCFTR-G542X mice and Cftr−/− hCFTR-G542X mice treated with an oral dose of 0.9 mg/ml PTC124.