ABCMdb

PMID: 18272502

Du M, Liu X, Welch EM, Hirawat S, Peltz SW, Bedwell DM
PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model.
Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):2064-9. Epub 2008 Feb 6., 2008-02-12 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly542* ABCC7 p.Gly542* 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. Login to comment 4 ABCC7 p.Gly542* 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. Login to comment 6 ABCC7 p.Gly542* These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. Login to comment 12 ABCC7 p.Gly542* Of these mutations, CFTR-G542X is the most common. Login to comment 24 ABCC7 p.Gly542* ABCC7 p.Gly542* Accordingly, in the present work we show that subcutaneous (s.c.) or oral administration of PTC124 suppressed the G542X mutation in a CF mouse model that expressed a human CFTR-G542X transgene in a Cftr-/- background, leading to a significant restoration of CFTR expression and function. Login to comment 26 ABCC7 p.Gly542* Results Subcutaneous Injection of PTC124 Suppresses Nonsense Mutations and Induces hCFTR Expression in Cftr-/- hCFTR-G542X Mice. Login to comment 27 ABCC7 p.Gly542* ABCC7 p.Gly542* We constructed a CF transgenic mouse model that expressed a human CFTR (hCFTR) cDNA containing the G542X premature stop mutation in a mouse line that carried a knockout of the endogenous Cftr locus (referred to hereafter as the Cftr-/- hCFTR-G542X mouse line) (8). Login to comment 38 ABCC7 p.Gly542* Because of these differences, we used the compact, intestine-specific rat fatty acid-binding protein (FABP) promoter to drive expression of the CFTR-G542X transgene. Login to comment 39 ABCC7 p.Gly542* ABCC7 p.Gly542* This Cftr-/- hCFTR-G542X mouse model was used to show that both gentamicin and amikacin could suppress the hCFTR-G542X mutation and partially restore CFTR protein expression and function (7, 8). Login to comment 40 ABCC7 p.Gly542* ABCC7 p.Gly542* To examine the ability of PTC124 to suppress the hCFTR-G542X mutation in vivo, once daily s.c. injections of PTC124 were administered to Cftr-/- hCFTR-G542X mice at dosages of 60, 30, or 15 mg/kg body weight for 14-21 days. Login to comment 45 ABCC7 p.Gly542* Similarly, no hCFTR protein was detected in intestinal tissues from untreated Cftr-/- hCFTR-G542X mice with hCFTR-specific antiserum. Login to comment 46 ABCC7 p.Gly542* However, strong hCFTR staining was observed at the apical surface of epithelial cells in submucosal glands from Cftr-/- hCFTR-G542X mice treated with 60 mg/kg PTC124 and, as observed, in tissues from mice treated with 34 mg/kg gentamicin. Login to comment 48 ABCC7 p.Gly542* ABCC7 p.Gly542* These results indicate that PTC124 can suppress the G542X mutation and partially restore hCFTR protein expression in Cftr-/- hCFTR-G542X mice. Login to comment 49 ABCC7 p.Gly542* Subcutaneous Injection of PTC124 Partially Restores cAMP-Stimulated Chloride Channel Activity in Cftr-/- hCFTR-G542X Mice. Login to comment 51 ABCC7 p.Gly542* We reported that cAMP-dependent transepithelial chloride conductance appeared in intestinal tissues of Cftr-/- hCFTR-G542X mice after treatment with 34 mg/kg gentamicin (7, 8). Login to comment 53 ABCC7 p.Gly542* ABCC7 p.Gly542* Supporting information (SI) Fig. 7 shows representative short-circuit current tracings obtained from Cftrϩ/ϩ mice, untreated Cftr-/- hCFTR-G542X mice, and Cftr-/- hCFTR-G542X mice treated with once daily s.c. injections of either 60 mg/kg PTC124 or 34 mg/kg gentamicin. Login to comment 54 ABCC7 p.Gly542* The intestinal tissues harvested from untreated Cftr-/- hCFTR-G542X mice showed no change in short-circuit currents after the addition of forskolin, a cAMP agonist. Login to comment 55 ABCC7 p.Gly542* In contrast, forskolin addition frequently induced an increase in short-circuit current in intestinal tissues from Cftr-/- hCFTR-G542X mice injected with 60 mg/kg PTC124 or 34 mg/kg gentamicin once a day for 14-21 days. Login to comment 56 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Fig. 2 summarizes the data collected from short-circuit current measurements from intestinal tissues harvested from untreated Cftr-/- hCFTR-G542X mice, Cftr-/- hCFTR-G542X mice treated with three different dosages of PTC124 for 14-21 days, or Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin for 14-21 days. Login to comment 58 ABCC7 p.Gly542* In untreated Cftr-/- hCFTR-G542X mice, we detected cAMP-stimulated short-circuit currents in only 8% of samples (1 of 12), resulting in an average current of 0.20 ␮A/cm2 . Login to comment 59 ABCC7 p.Gly542* In the Cftr-/- hCFTR-G542X mice treated with 60 mg/kg PTC124, 47% of samples (8 of 17) showed a positive reaction after the addition of forskolin, resulting in an average current of 1.66 ␮A/cm2 . Login to comment 60 ABCC7 p.Gly542* Similarly, Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin manifested cAMP-stimulated short-circuit currents in 63% of samples (5 of 8), resulting in an average current of 1.67 ␮A/cm2 . Login to comment 62 ABCC7 p.Gly542* ABCC7 p.Gly542* 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. Login to comment 63 ABCC7 p.Gly542* 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. Login to comment 67 ABCC7 p.Gly542* PTC124 to Cftr-/- hCFTR-G542X mice once daily by s.c. injection produced a statistically significant increase (P Ͻ 0.05) in cAMP-stimulated transepithelial chloride currents in intestinal tissues relative to untreated controls, resulting in 29% of the mean cAMP-stimulated short currents measured in wild-type mice. Login to comment 69 ABCC7 p.Gly542* The s.c. administration of PTC124 at 30 or 15 mg/kg once daily did not show a significant increase in cAMP-stimulated short currents after forskolin addition relative to untreated Cftr-/- hCFTR-G542X mice, indicating that 60 mg/kg PTC124 is the minimal effective dose with this administration protocol. Login to comment 73 ABCC7 p.Gly542* Oral Administration of PTC124 Partially Restores hCFTR Protein Expression and cAMP-Stimulated Chloride Channel Activity in Cftr-/- hCFTR-G542X Mice. Login to comment 74 ABCC7 p.Gly542* ABCC7 p.Gly542* The results above indicated that once daily s.c. injection of 60 mg/kg PTC124 suppressed the G542X mutation in Cftr-/- hCFTR-G542X mice and restored the expression of functional hCFTR protein. Login to comment 75 ABCC7 p.Gly542* To determine serum levels resulting from this administration protocol, age-matched Cftrϩ/- hCFTR-G542X mice were injected with 60 mg/kg PTC124, and orbital blood was collected at various times after injection (Fig. 3A). Login to comment 78 ABCC7 p.Gly542* Because of the potential for intestinal blockage in Cftr-/- hCFTR-G542X mice, these animals are routinely fed a liquid complete diet (Peptamen Complete Elemental Diet; Nestle´) upon weaning to promote survival. Login to comment 79 ABCC7 p.Gly542* To identify an oral dose of PTC124 that might provide a therapeutic benefit, the compound was dissolved in the liquid diet at two concentrations (0.3 and 0.9 mg/ml) and given to Cftrϩ/- hCFTR-G542X mice as the sole source of food and water for 2-6 days. Login to comment 83 ABCC7 p.Gly542* ABCC7 p.Gly542* Because the average serum levels obtained by oral administration with these doses of PTC124 effectively bracketed the peak serum levels obtained immediately after s.c. injection and the optimal dosing established in the mdx studies, Cftr-/- hCFTR-G542X mice were fed the Peptamen liquid diet containing 0.3 or 0.9 mg/ml PTC124 for 14-21 days to determine whether this dosing protocol could lead to effective suppression of the G542X mutation. Login to comment 86 ABCC7 p.Gly542* In mice maintained on Cftr-/- hCFTR-G542X Cftr+/+ UnTreated Gent 34mg/kg PTC124 60mg/kg PTC124 30mg/kg PTC124 15mg/kg UnTreated PTC124 60mg/kg Positive/Total Reactions 1/12 5/8 8/17 2/16 2/13 12/12 10/10 100% 5.78 0.48 101% Positive Reactions (%) 8% 63% 47% 13% 15% 100% Mean Current (µA/cm2 ) 0.20 1.67 1.66 0.29 0.12 5.72 P value - 0.023 0.034 0.36 0.36 - % WT Current 3.5% 29% 29% 5.1% 2.1% 100% ShortCircuitCurrent(µA/cm2 ) 0 2 4 6 8 10 12 14 Fig. 2. Login to comment 87 ABCC7 p.Gly542* Effect of PTC124 s.c. injection on cAMP-stimulated transepithelial chloride currents in intestinal tissues from Cftr-/- hCFTR-G542X and Cftrϩ/ϩ mice. Login to comment 93 ABCC7 p.Gly542* No hCFTR protein was detected in the intestines of treated Cftr-/- hCFTR-G542X mice with preimmune serum. Login to comment 95 ABCC7 p.Gly542* These results indicated that oral administration of PTC124 could partially restore hCFTR protein expression in Cftr-/- hCFTR-G542X mice. Login to comment 97 ABCC7 p.Gly542* We found that intestinal tissues harvested from Cftr-/- hCFTR-G542X mice fed the liquid diet with 0.3 mg/ml PTC124 yielded cAMP-stimulated short-circuit currents in 29% of intestinal tissues assayed (7 of 24), resulting in an average currents of 0.91 ␮A/cm2 . Login to comment 100 ABCC7 p.Gly542* The mean cAMP-stimulated short-circuit currents observed in Cftr-/- hCFTR-G542X mice fed the liquid diet with 0.9 mg/ml PTC124 was 24% of the average cAMP-stimulated currents measured in wild-type mice. Login to comment 104 ABCC7 p.Gly542* Finally, cAMP-stimulated short-circuit currents were not observed in Cftr-/- knockout mice that lacked the hCFTR-G542X transgene, regardless of whether they were untreated or treated with 0.9 mg/ml PTC124. Login to comment 105 ABCC7 p.Gly542* This result confirmed that the CFTR function observed after PTC124 treatment depends on the presence of the hCFTR-G542X transgene, consistent with a role for PTC124 in inducing readthrough of premature translation termination codons. Login to comment 108 ABCC7 p.Gly542* Hence, although PTC124 is thought to have a direct effect on nonsense codon readthrough (14), it is formally possible that the ability of the drug to suppress the hCFTR-G542X mutation is attributable to an effect on mRNA stability. Login to comment 109 ABCC7 p.Gly542* However, RT-PCR analysis demonstrated that there were comparable levels of mRNA from the hCFTR-G542X transgene in untreated mice and in mice treated with 0.9 mg/ml PTC124 in the liquid diet (Fig. 6). Login to comment 118 ABCC7 p.Gly542* (A) Blood was taken from different age-matched Cftrϩ/- hCFTR-G542X mice at the indicated times after s.c. injection of 60 mg/kg PTC124. Login to comment 119 ABCC7 p.Gly542* ABCC7 p.Gly542* (B) Blood samples were taken after feeding Cftrϩ/- 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. Login to comment 123 ABCC7 p.Gly542* 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. Login to comment 126 ABCC7 p.Gly542* to Cftr-/- hCFTR-G542X mice by s.c. injection restored 29% of the normal intestinal transepithelial cAMP-stimulated short-circuit currents observed in Cftrϩ/ϩ mice. Login to comment 129 ABCC7 p.Gly542* Our data indicate that the human CFTR protein produced by suppression of the nonsense mutation in Cftr-/- hCFTR-G542X mice is located primarily at the epithelial surface of the submucosal glands in the duodenum. Login to comment 131 ABCC7 p.Gly542* To determine whether the suppressed level of CFTR expression can prevent the frequent intestinal blockage associated with the lack of functional CFTR protein, we are currently making a knockin Cftr-G542X mouse that will have a normal distribution of CFTR expression. Login to comment 132 ABCC7 p.Gly542* We observed an occasional weak cAMP-stimulated current in intestinal tissues from Cftr-/- hCFTR-G542X mice in the absence of any treatment but not in Cftr-/- knockout mice that lacked the transgene (see Fig. 5). Login to comment 133 ABCC7 p.Gly542* ABCC7 p.Gly542* These data suggest that there may be a low level of endogenous readthrough of the premature stop codon in Cftr-/- hCFTR-G542X mice that we occasionally detect because such residual activity would not be observed in Cftr-/- knockout mice that lack the hCFTR-G542X transgene. Login to comment 134 ABCC7 p.Gly542* Previous studies have shown that some nonsense codons (particularly UGA codons like that encoded by the G542X mutation) have a higher basal level of readthrough and are more susceptible to readthrough induced by aminoglycosides than other stop codons (19-21). Login to comment 140 ABCC7 p.Gly542* ABCC7 p.Gly542* In conclusion, our results demonstrate that PTC124 induces readthrough of the premature translation termination codon encoded by the hCFTR-G542X nonsense mutation, resulting in the partial restoration of CFTR protein and cAMP-activated chloride currents in the intestines of Cftr-/- hCFTR-G542X transgenic mice. Login to comment 142 ABCC7 p.Gly542* Cftr-/- hCFTR-G542X Cftr+/+ Cftr-/- UnTreated PTC124 0.3mg/ml PTC124 0.9mg/ml UnTreated PTC124 0.9mg/ml UnTreated PTC124 0.9mg/ml 0/8 0/8 0% 0.00 - 0% 0% 0.00 - Positive/Total Reactions 2/16 7/24 5/11 12/12 0% 13/13 100% 6.15 0.35 108% Positive Reactions (%) 13% 29% 45% 100% Mean Current (µA/cm2) 0.20 0.91 1.35 5.72 P value - 0.021 0.027 - % WT Current 3.5% 16% 24% 100% ShortCircuitCurrent(µA/cm2 ) 0 4 8 12 16 ++++++++ 2 6 10 14 Fig. 5. Login to comment 143 ABCC7 p.Gly542* Effect of PTC124 oral administration on cAMP-stimulated transepithelial chloride currents in intestinal tissues from Cftr-/- hCFTR-G542X, Cftrϩ/ϩ, and Cftr-/- mice. Login to comment 147 ABCC7 p.Gly542* ABCC7 p.Gly542* 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. Login to comment 149 ABCC7 p.Gly542* The rat FABP promoter was used to drive expression of the hCFTR transgene that contained the G542X (UGA) premature stop mutation. Login to comment 150 ABCC7 p.Gly542* ABCC7 p.Gly542* The plasmid construction of the FABP-hCFTR-G542X transgene and generation of the Cftr-/- hCFTR-G542X mouse were described in refs. 7 and 8. Login to comment 153 ABCC7 p.Gly542* Subcutaneous injections with the indicated doses of PTC124 or gentamicin were made in the hind limb of age-matched Cftr-/- hCFTR-G542X mice. Login to comment 156 ABCC7 p.Gly542* Because of the potential for intestinal blockage in Cftr-/- hCFTR-G542X mice, they were maintained on a liquid diet (Peptamen) after weaning, and other food and water were withheld. Login to comment 190 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* To monitor expression of the hCFTR-G542X transgene, mRNA was isolated from intestinal tissues of untreated Cftr-/- hCFTR-G542X mice and Cftr-/- hCFTR-G542X mice treated with 0.9 mg/ml PTC124 in the liquid diet for 17 days. Login to comment 191 ABCC7 p.Gly542* RT-PCR analysis of hCFTR-G542X mRNA was done by using a SuperScript III one-step RT-PCR system with platinum Taq polymerase (Invitrogen). Login to comment 193 ABCC7 p.Gly542* The primers used to amplify a 1,557-bp fragment from the hCFTR-G542X mRNA were DB985 (5Ј-CAAGATAGAA AGAGGACAGT TGTT-3Ј) and DB986 (5Ј-TTGAGGGTTG ACATAGGTGC TTGAA-3Ј). Login to comment 217 ABCC7 p.Gly542* Du M, et al. (2006) Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model. Login to comment 220 ABCC7 p.Gly542* Du M, et al. (2002) Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment