ABCMdb

PMID: 21779978

Rowe SM, Sloane P, Tang LP, Backer K, Mazur M, Buckley-Lanier J, Nudelman I, Belakhov V, Bebok Z, Schwiebert E, Baasov T, Bedwell DM
Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54.
J Mol Med (Berl). 2011 Jul 22., 2011-07-22 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Trp1282* In a fluorescence-based halide efflux assay, NB54 partially restored halide efflux in a CF bronchial epithelial cell line (CFTR genotype W1282X/F508del), but not in a CF epithelial cell line lacking a PTC (F508del/F508del). Login to comment 5 ABCC7 p.Trp1282* ABCC7 p.Gly542* In polarized airway epithelial cells expressing either a CFTR-W1282X or - G542X cDNA, treatment with NB54 increased stimulated short-circuit current (ISC) with greater efficiency than gentamicin. Login to comment 6 ABCC7 p.Gly542* NB54 and gentamicin induced comparable increases in forskolin-stimulated ISC in primary airway epithelial cells derived from a G542X/F508del CF donor. Login to comment 7 ABCC7 p.Gly542* Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents Electronic supplementary material The online version of this article (doi:10.1007/s00109-011-0787-6) contains supplementary material, which is available to authorized users. Login to comment 32 ABCC7 p.Gly542* We also show that NB54 restores a comparable level of CFTR expression and function as gentamicin in transgenic mice expressing a human CFTR-G542X cDNA. Login to comment 39 ABCC7 p.Trp1282* Growth of stable cell lines expressing recombinant CFTR CFTR-W1282X cDNA were stably transfected into HeLa and CFBE41o-cells using the TranzVector™ (Tranzyme, Inc., Birmingham, AL) as described [22]. Login to comment 43 ABCC7 p.Gly542* Primary human airway epithelial cells were derived from lung explants of CF subjects who provided written informed consent and were confirmed to harbor two severe CFTR mutations (including the G542X premature termination codon) by methods described previously [24, 25]. Login to comment 46 ABCC7 p.Trp1282* SPQ studies of halide efflux in heterologous cells HeLa cells stably transfected with a lentiviral system carrying a CFTR-W1282X cDNA were studied with the halide quenched dye 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ, Molecular Probes Inc., Eugene, OR) as described by the manufacturer and previously published [23, 26, 27]. Login to comment 65 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Mouse lines and treatment protocols The CFTR-G542X mice used in this study contained the Cftrtm1Cam knockout [30] and expressed a human CFTR transgene with the G542X premature stop mutation [17, 31, 32] (referred to as Cftr-/- hCFTR-G542X mice). Login to comment 78 ABCC7 p.Trp1282* Results Translational readthrough screen of synthetic aminoglycosides in human epithelial cell lines To determine whether the synthetic aminoglycosides NB30 and NB54 induce enough translational readthrough of CFTR PTCs to partially restore CFTR function using a stimulated halide transport assay, we first tested the compounds using HeLa cells stably transduced with a lentiviral system driving CFTR-W1282X expression. Login to comment 82 ABCC7 p.Trp1282* To confirm the effects of aminoglycosides on readthrough in a more physiologically relevant cell type with endogenous CFTR expression, we next examined the IB3-1 bronchial epithelial cell line (CFTR genotype W1282X/ F508del) in comparison to a CF epithelial cell line derived from nasal polyps from a homozygous CFTR-F508del donor (CFNPE cells). Login to comment 93 ABCC7 p.Trp1282* Fig. 2 Fluorescence-based halide efflux assay of cells expressing CFTR-W1282X following treatment with gentamicin, NB30, or NB54. Login to comment 94 ABCC7 p.Trp1282* a HeLa cells expressing CFTR-W1282X were grown on glass coverslips were treated with 500 μg/ml of the indicated aminoglycoside (gentamicin, NB30, or NB54) for 24 h, or mock treated with vehicle, and studied by fluorescence-based halide efflux (SPQ). Login to comment 99 ABCC7 p.Trp1282* b IB3-1 cells (endogenous CFTR genotype W1282X/F508Del) were incubated with the aminoglycosides NB30, NB54, or gentamicin at the concentrations indicated for 24 h, and halide efflux was quantified by percentage change in fluorescence over basal compared to control following activation with forskolin (20 μM) and genistein (50 μM). Login to comment 105 ABCC7 p.Trp1282* ABCC7 p.Trp1282* In CFBE41o-cells stably transduced with a CFTR-W1282X cDNA, NB54 treatment (500 μg/ml) increased ISC 0.38 μA/cm2 more than in cells treated with vehicle alone. Login to comment 107 ABCC7 p.Gly542* ABCC7 p.Gly542* To test the more common CFTR-G542X allele and examine dose dependence, CFBE41o- human airway cells transduced with an adenovirus vector expressing a CFTR-G542X cDNA were tested following treatment with gentamicin or NB54. Login to comment 116 ABCC7 p.Trp1282* ABCC7 p.Gly542* We obtained primary HBE cells from a compound heterozygote subject (G542X/F508del) following a lung Fig. 3 Increased W1282X-CFTR dependent short-circuit current following incubation of synthetic aminoglycosides. Login to comment 117 ABCC7 p.Trp1282* a Representative short-circuit current tracings obtained from CFBE41o- expressing W1282X CFTR mounted in modified Ussing chambers and studied under voltage clamp conditions. Login to comment 122 ABCC7 p.Gly542* ABCC7 p.Gly542* *P<0.05; **P<0.005; ±SEM, n=16 Fig. 4 Short-circuit current assay of CFBE41o-cells transduced with an adenovirus expressing CFTR-G542X following treatment with gentamicin, NB30, or NB54. Login to comment 123 ABCC7 p.Gly542* ABCC7 p.Gly542* a Representative short-circuit current tracings of CFBE41o-cells were grown on air-liquid interface, transduced with adenovirus encoding CFTR-G542X, allowed to recover 48 h, and exposed to aminoglycosides (500 μg/ml) for 24 h. Login to comment 127 ABCC7 p.Gly542* ABCC7 p.Gly542* b Mean stimulated short-circuit currents measured in CFBE41o-cells transduced with adenovirus CFTR-G542X following treatment with the indicated doses of gentamicin or NB54. Login to comment 142 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Readthrough of CFTR-G542X in vivo induced by the synthetic aminoglycoside NB54 To examine whether NB54 could suppress a CFTR PTC in vivo, we next utilized a transgenic human CFTR-G542X (hCFTR-G542X) mouse line [17, 31-33]. Login to comment 143 ABCC7 p.Gly542* In this mouse model, the hCFTR-G542X transgene is expressed from the rat intestinal FABP promoter in a mouse Cftr-/- background. Login to comment 144 ABCC7 p.Gly542* Samples from the ileum of untreated Cftr+/+ littermates carrying the transgene were used as positive controls (Fig. 6a), while corresponding samples from untreated Cftr-/- mice carrying the hCFTR-G542X transgene were used as negative controls (Fig. 6b). Login to comment 146 ABCC7 p.Gly542* Fully differentiated primary airway cells derived from a CF (G542X/ F508Del) donor were grown at air-liquid interface until terminally differentiated (e.g., 90% ciliated), and then treated with NB54, gentamicin, or vehicle (500 μg/ml) for the times indicated, then mounted in modified Ussing chambers under voltage clamp conditions. Login to comment 150 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Representative short-circuit current tracings are shown from a untreated Cftr+/+ hCFTR-G542X mice (WT control), b untreated Cftr-/- hCFTR-G542X (negative control), c gentamicin-treated Cftr-/- hCFTR-G542X (60 mg/kg), and d NB54-treated Cftr-/- hCFTR-G542X (120 mg/kg). Login to comment 151 ABCC7 p.Gly542* e Scatter plot of the total ISC data from Cftr-/- hCFTR-G542X mice (untreated, gentamicin treated, and NB54 treated) that combines the forskolin/IBMX and carbachol responses. Login to comment 156 ABCC7 p.Gly542* ABCC7 p.Gly542* A scatter plot of the total ISC (ΔISC following forskolin/ IBMX + carbachol) from all Cftr-/- hCFTR-G542X mice (untreated, gentamicin treated, and NB54 treated) is presented in Fig. 6e, while an analysis of that data is represented in Fig. 6f. In Cftr+/+ hCFTR-G542X mice, ileal tissues yielded a mean cAMP-stimulated ISC of 161 μA/cm2 10 min after forskolin/IBMX addition, and a further increase in ISC of 209 μA/cm2 following the addition of carbachol (a muscarinic agonist that transiently enhances the cAMP-dependent chloride secretory response in intestinal tissues by activating Ca2+ -dependent potassium channels in the basolateral plasma membrane [37]). Login to comment 158 ABCC7 p.Gly542* In contrast, stimulated responses were effectively absent in untreated Cftr-/- hCFTR-G542X control mice. Login to comment 161 ABCC7 p.Gly542* We initially compared the relative efficacy of gentamicin and NB54 to restore activated ISC, a measure of full-length CFTR protein function resulting from suppression of the hCFTR-G542X mutation. Login to comment 169 ABCC7 p.Gly542* Next, we examined the ability of NB54 to induce readthrough and restore CFTR activity in the Cftr-/- hCFTR-G542X transgenic mouse model. Login to comment 176 ABCC7 p.Gly542* Only background staining was observed in untreated Cftr-/- hCFTR-G542X mice (Fig. 7a). Login to comment 193 ABCC7 p.Trp1282* ABCC7 p.Gly542* We tested the ability of these compounds to suppress two CFTR PTCs (G542X and W1282X) in a variety of CF cellular models, including heterologous CFTR expression systems, immortalized CF cell lines, and primary HBE cells isolated from a CF lung. Login to comment 194 ABCC7 p.Gly542* In addition, we examined the ability of these compounds to suppress the human CFTR-G542X mutation in a transgenic mouse model. Login to comment 197 ABCC7 p.Trp1282* ABCC7 p.Trp1282* We found that NB30 and NB54 treatment restored a level of cAMP-stimulated halide transport that was comparable to gentamicin in both HeLa cells stably transduced with a lentiviral system expressing CFTR-W1282X and in the IB3-1 bronchial epithelial cell line (W1282X/F508del, Fig. 2). Login to comment 199 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Gly542* ABCC7 p.Gly542* The maximal ISC measured in CFBE41o-airway epithelial monolayers stably transduced with a lentivirus vector expressing CFTR-W1282X or an adenovirus vector expressing CFTR-G542X was consistently higher with NB54 than gentamicin, and NB54 was also nontoxic at these doses (Figs. Login to comment 201 ABCC7 p.Gly542* NB54 also induced a cAMP-stimulated ISC that was comparable to gentamicin in primary HBE cells carrying the CFTR-G542X mutation, and the induction of that response occurred sooner than we observed with gentamicin treatment (Fig. 5). Login to comment 208 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* a Samples from untreated Cftr-/- hCFTR-G542X, b samples from Cftr-/- hCFTR-G542X mice treated with 60 mg/kg gentamicin, and c samples from Cftr-/- hCFTR-G542X mice treated with 120 mg/kg NB54. Login to comment 213 ABCC7 p.Gly542* As a further test of the ability of NB54 to suppress CF-related PTCs, we examined the propensity of NB54 to restore human CFTR expression in Cftr-/- hCFTR-G542X transgenic mice (Fig. 6). Login to comment 214 ABCC7 p.Gly542* We found that NB54 induced significant and dose-dependent increases in stimulated ISC in Cftr-/- hCFTR-G542X transgenic mice, resulting in 15.8% of the current measured in WT mice at a dose of 120 mg/kg. Login to comment 218 ABCC7 p.Gly542* Since it was demonstrated in a previous study that gentamicin and PTC124 (ataluren) restored a comparable level of CFTR activity in the same hCFTR-G542X Cftr-/- mouse model [17], the level of CFTR function restored by NB54 should be similar to PTC124 as well. Login to comment 238 ABCC7 p.Trp1282* Shoshani T, Kerem E, Szeinberg A, Augarten A, Yahav Y, Cohen D, Rivlin J, Tal A, Kerem B (1994) Similar levels of mRNA from the W1282X and the delta F508 cystic fibrosis alleles, in nasal epithelial cells. Login to comment 261 ABCC7 p.Gly542* Am J Respir Cell Mol Biol 37:57-66 17. Du M, Liu X, Welch EM, Hirawat S, Peltz SW, Bedwell DM (2008) PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment 272 ABCC7 p.Trp1282* Mol Ther 2:47-55 23. Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP (2007) Restoration of W1282X CFTR activity by enhanced expression. Login to comment 285 ABCC7 p.Gly542* Nat Genet 4:35-41 31. Du M, Jones JR, Lanier J, Keeling KM, Lindsey RJ, Tousson A, Bebok Z, Whitsett JA, Dey CR, Colledge WH et al (2002) Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment 286 ABCC7 p.Gly542* ABCC7 p.Gly542* J Mol Med 80:595-604 32. Du M, Keeling KM, Fan L, Liu X, Kovacs T, Sorscher E, Bedwell DM (2006) Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model. J Mol Med 84:573-582 33. Du M, Keeling KM, Fan L, Liu X, Bedwell DM (2009) Poly-L-aspartic acid enhances and prolongs gentamicin-mediated suppression of the CFTR-G542X mutation in a cystic fibrosis mouse model. J Biol Chem 284:6885-6892 34. Login to comment