ABCMdb

PMID: 24251786

Xue X, Mutyam V, Tang L, Biswas S, Du M, Jackson LA, Dai Y, Belakhov V, Shalev M, Chen F, Schacht J, J Bridges R, Baasov T, Hong J, Bedwell DM, Rowe SM
Synthetic aminoglycosides efficiently suppress cystic fibrosis transmembrane conductance regulator nonsense mutations and are enhanced by ivacaftor.
Am J Respir Cell Mol Biol. 2014 Apr;50(4):805-16. doi: 10.1165/rcmb.2013-0282OC., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Arg1162* Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. Login to comment 3 ABCC7 p.Gly542* NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/ delF508), a highly relevant preclinical model with endogenous CFTR expression. Login to comment 5 ABCC7 p.Gly542* NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. Login to comment 28 ABCC7 p.Gly542* For example, poly-L-aspartic acid, a compound previously shown to significantly reduce aminoglycoside toxicity, was found to increase both the level and duration of readthrough in a CFTR-G542X transgenic mouse (13). Login to comment 34 ABCC7 p.Gly542* One of the early compounds produced, NB54, suppressed PTCs to a level comparable to gentamicin in immortalized and primary human CF cells, as well as in the CFTR-G542X transgenic mouse model, while exhibiting low toxicity, suggesting a potential advantage of this approach (21). Login to comment 43 ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg1162* Dual Luciferase Assay Readthrough cassettes contained the G542X, R553X, R1162X, or W1282X CFTR PTCs (or the corresponding wild-type codon) together with three codons of upstream and downstream human CFTR sequence (Figure 2A; see also Table E1 in the online supplement). Login to comment 62 ABCC7 p.Gly542* Cftr knockout mice expressing a human CFTR-G542X transgene under intestine-specific rat fatty acid binding protein were treated by subcutaneous injection once daily for 14 days (6, 13, 15). Login to comment 76 ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg1162* To test suppression of the four most common CFTR PTCs (G542X, R553X, R1162X, and W1282X), we constructed dual-luciferase reporters that each contained a Renilla gene, a firefly gene, and a CFTR readthrough cassette with each PTC, and the context of three additional codons on either side of the PTC (Figure 2A). Login to comment 95 ABCC7 p.Gly542* Initially, we treated the CFTR-corrected CFBE41o2 cells expressing the G542X dual-luciferase reporter with NB124. Login to comment 98 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Qualitatively similar results were obtained for the R1162X and W1282X PTCs (Figures 2D and 2E). Login to comment 99 ABCC7 p.Trp1282* ABCC7 p.Arg1162* NB124 (100 mM) induced a 2.5-fold increase in readthrough at the highest dose tested with the R1162X readthrough reporter (Figure 2D), and a 1.9-fold increase in readthrough with the W1282X construct. Login to comment 101 ABCC7 p.Arg553* In contrast, NB124 induced a much lower (1.2-fold) level of readthrough at the R553X PTC (Figure 2C), which was slightly lower than the readthrough induced by gentamicin. Login to comment 103 ABCC7 p.Arg553* Furthermore, because all four of the CFTR PTCs tested were UGA nonsense codons, the differences observed in the readthrough pattern observed with the R553X mutation compared with the other PTCs demonstrates that the surrounding sequence context has a significant influence on the level of nonsense suppression obtained. Login to comment 108 ABCC7 p.Gly542* To assess the efficacy of synthetic aminoglycosides, FRT-G542X cells were treated for 48 hours, then CFTR activity determined by the change in Gt after forskolin (10 mM) activation. Login to comment 115 ABCC7 p.Gly542* When taken together, these results indicated that NB124 suppresses the relatively common CFTR-G542X mutation more efficiently than other tested aminoglycosides. Login to comment 119 ABCC7 p.Gly542* We therefore next used CFTR functional assays on primary HBE cells (CFTR genotype G542X/ delF508) to evaluate the PTC suppression efficacy of synthetic aminoglycosides. Login to comment 124 ABCC7 p.Gly542* ABCC7 p.Gly542* Partial Restoration of CFTR Function in Cftr2/2 Mice Expressing a Human CFTR-G542X Transgene The in vitro results described previously here demonstrate that several synthetic aminoglycosides suppress CFTR PTCs more effectively than gentamicin, with NB124 most effectively restoring CFTR function across several clinically relevant alleles, including CFTR-G542X, the most common disease-causing nonsense allele. Login to comment 125 ABCC7 p.Gly542* ABCC7 p.Gly542* Based on those results, we next tested the ability of NB124 to suppress CFTR in a Cftr knockout mouse line expressing a human CFTR-G542X transgene under control of the intestine-specific rat fatty acid-binding protein promoter (referred to hereafter as Cftr2/2 hCFTR-G542X). Login to comment 128 ABCC7 p.Gly542* ABCC7 p.Gly542* Figures 5A and 5B show representative Isc tracings from Cftr1/1 hCFTR-G542X and Cftr2/2 hCFTR-G542X mice (positive and Figure 3. Login to comment 129 ABCC7 p.Gly542* Improved CFTR function and expression in CFTR-G542X-expressing Fischer rat thyroid (FRT) monolayers. Login to comment 133 ABCC7 p.Gly542* *P , 0.05, ***P , 0.001, ****P , 0.0001 versus vehicle or gentamicin negative controls, respectively), and Figures 5C and 5D show Isc tracings from Cftr2/2 hCFTR-G542X mice treated with 60 mg/kg gentamicin and 30 mg/kg NB124, respectively. Login to comment 134 ABCC7 p.Gly542* These data are summarized in Figure 5E, which shows a scatter plot of forskolin-stimulated Isc from ileum samples obtained from untreated and treated Cftr2/2 hCFTR-G542X mice. Login to comment 135 ABCC7 p.Gly542* The mean forskolin-stimulated Isc was 5.7 (6 3.7) mA/cm2 in ileum samples from untreated Cftr2/2 hCFTR-G542X mice (negative controls), and 255.7 (6 36.1) mA/cm2 in samples from wild-type mice (positive controls). Login to comment 136 ABCC7 p.Gly542* In Cftr2/2 hCFTR-G542X mice treated with 30 mg/kg gentamicin, we did not observe a significant increase in Isc relative to the untreated controls (5.3 6 4.1 mA/cm2 ). Login to comment 138 ABCC7 p.Gly542* In contrast, significant increases in forskolin-stimulated Isc were observed in Cftr2/2 hCFTR-G542X mice treated with 30 and 60 mg/kg NB124 (14.7 6 7.8 and 12.4 6 9.2, respectively). Login to comment 141 ABCC7 p.Gly542* When taken together, these results indicate that NB124 restores greater CFTR function (and at a 2-fold lower dose) than gentamicin in Cftr2/2 hCFTR-G542X transgenic mice, and has a slightly more favorable dose-response relationship. Login to comment 152 ABCC7 p.Gly542* Because reliable readthrough assays in primary HBE cell monolayers and in vivo found that NB124 suppresses the G542X-CFTR nonsense mutation roughly 2.5-fold more effectively than gentamicin in vivo, these results indicate that the efficacy:toxicity ratio of NB124 is approximately 10-fold better than gentamicin. Login to comment 156 ABCC7 p.Gly542* Restoration of CFTR function in CF primary human bronchial epithelial (HBE) cells derived from a G542X/F508del donor. Login to comment 164 ABCC7 p.Arg1162* cells expressing theCFTR-R1162X allele. Login to comment 170 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Representative tracings of (A) untreated Cftr1/1 hCFTR-G542X mice (WT control), (B) untreated Cftr2/2 hCFTR-G542X (negative control), (C) gentamicin-treated Cftr2/2 hCFTR-G542X (60 mg/ kg), and (D) NB124-treated Cftr2/2 hCFTR-G542X (30 mg/kg). Login to comment 171 ABCC7 p.Gly542* (E) Scatter plot of all data from Cftr2/2 hCFTR-G542X mice (untreated, gentamicin treated, and NB124 treated). Login to comment 177 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Representative images are shown from (F) untreated Cftr2/2 hCFTR-G542X (negative control), (G) gentamicin-treated Cftr2/2 hCFTR-G542X (60 mg/kg), and (H) NB124-treated Cftr2/2 hCFTR-G542X (30 mg/kg). Login to comment 193 ABCC7 p.Trp1282* ABCC7 p.Gly542* ABCC7 p.Arg1162* We found that three CFTR UGA mutations (G542X, R1162X, and W1282X) exhibited qualitatively similar responses to the compounds and showed maximal suppression with NB124. Login to comment 194 ABCC7 p.Arg553* In contrast, the UGA mutation associated with R553X was more refractory to suppression, demonstrating that sequence context plays an important role in PTC suppression. Further studies are needed to better understand how the sequence context causes these differences in PTC suppression. Login to comment 199 ABCC7 p.Arg1162* The immortalized CFBE41o2 cells stably transfected with CFTR-R1162X or CFTRW1282X cDNAs under cytomegalovirus Table 1: Serum Levels of Gentamicin and NB124 Serum Level Dose (mg/ml) Compound (mg/kg) 20 min after Injection 120 min after Injection Gentamicin 30 42.6 6 0.4 19.9 6 1.3 NB124 30 50.2 6 1.1* 7.0 6 0.4ߤ Gentamicin 60 64.2 6 1.0 15.5 6 1.0 NB124 60 103.8 6 3.4ߤ 9.6 6 0.5* *P , 005 versus same dose of gentamicin at the same time point. Login to comment 216 ABCC7 p.Gly542* Finally, we used a mouse model that expressed a human CFTR-G542X transgene in a Cftr knockout background to compare the functional results obtained with NB124 and gentamicin (Figure 5). Login to comment 223 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (A and B) CFBE41o2 cells stably expressing CFTR-R1162X (A) or W1282X (B) were incubated with synthetic aminoglycosides (250 mg/ml) for 48 hours, followed by Isc measurements in Ussing chambers. Login to comment 230 ABCC7 p.Gly551Asp Recently, the CFTR potentiator, ivacaftor, was approved for clinical use for the treatment of patients with CF carrying the CFTR-G551D mutation (34). Login to comment