ABCMdb

PMID: 16541275

Du M, Keeling KM, Fan L, Liu X, Kovacs T, Sorscher E, Bedwell DM
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 (Berl). 2006 Jul;84(7):573-82. Epub 2006 Mar 16., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Gly542* David M. Bedwell Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model Received: 31 October 2005 / Accepted: 9 January 2006 / Published online: 16 March 2006 # Springer-Verlag 2006 Abstract Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease cystic fibrosis. Login to comment 7 ABCC7 p.Gly542* ABCC7 p.Gly542* We previously reported that gentamicin administration suppressed a CFTR premature stop mutation in a Cftr-/- mouse model carrying a human CFTR-G542X (hCFTR-G542X) transgene, resulting in the appearance of hCFTR protein and function. Login to comment 9 ABCC7 p.Gly542* ABCC7 p.Gly542* We then asked whether these doses could suppress the hCFTR-G542X mutation in the Cftr-/- hCFTR-G542X mouse model. Login to comment 11 ABCC7 p.Gly542* However, we found that amikacin suppressed the hCFTR-G542X premature stop mutation more effectively than gentamicin when administered at these clinically relevant doses. Login to comment 42 ABCC7 p.Gly542* We previously reported that the aminoglycoside gentamicin is capable of suppressing CFTR premature stop mutations in a CF transgenic mouse model, in which an hCFTR cDNA containing the G542X premature stop mutation was expressed under the control of the rat intestinal fatty acid binding protein (FABP) promoter in a Cftr-/- mouse. Login to comment 46 ABCC7 p.Gly542* ABCC7 p.Gly542* This hCFTR-G542X Cftr-/- mouse was used to show that once daily administration of 34 mg/kg of gentamicin or tobramycin via subcutaneous injections could suppress the G542X nonsense mutation and partially restore expression of functional hCFTR protein [20]. Login to comment 50 ABCC7 p.Gly542* In a previous study, we showed that gentamicin suppressed the hCFTR-G542X nonsense mutation in vivo, although peak serum concentrations used were well above the levels allowed during the recommended clinical use of these compounds [20]. Login to comment 52 ABCC7 p.Gly542* In the current study, we examined whether gentamicin and amikacin could suppress the G542X mutation when administered at doses within the recommended therapeutic range of serum peak and trough levels. Login to comment 53 ABCC7 p.Gly542* We obtained evidence that both compounds could suppress the hCFTR-G542X mutation in the Cftr-/- mouse model and partially restore functional CFTR expression when administered at doses that produced serum levels in the recommended range. Login to comment 54 ABCC7 p.Gly542* ABCC7 p.Gly542* However, our results indicate that amikacin suppressed the hCFTR-G542X mutation in vivo much more effectively than gentamicin under these conditions, suggesting that it may represent a better choice for the suppression of the hCFTR-G542X stop mutation (and possibly other premature stop mutations that cause CF). Login to comment 55 ABCC7 p.Gly542* Materials and methods Plasmid construction and generation of transgenic mice Expression of the hCFTR cDNA transgene that contained the G542X (UGA) premature stop mutation was driven by the rat FABP promoter. Login to comment 56 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* The plasmid construction of the FABP-hCFTR-G542X transgene and generation of the Cftr-/- hCFTR-G542X were described previously [20]. Login to comment 57 ABCC7 p.Gly542* In vitro translation system A modified form of a readthrough reporter system, previously used to monitor the suppression of stop mutations by aminoglycosides [4, 5], was used to determine whether amikacin (Calbiochem) could suppress the hCFTR-G542X mutation in an in vitro rabbit reticulocyte lysate (RRL) translation system (Promega TNT system). Login to comment 60 ABCC7 p.Gly542* In the present study, the readthrough cassette contained the hCFTR-G542X premature stop codon and six upstream and downstream codons from the hCFTR gene. Login to comment 64 ABCC7 p.Gly542* Efficient translation termination at the G542X stop mutation produced a 25-kDa protein, while suppression of the stop codon resulted in the synthesis of a 35-kDa protein. Login to comment 66 ABCC7 p.Gly542* ABCC7 p.Gly542* Suppression of the G542X mutation was expressed as percent readthrough, which was calculated as the 35 kda protein= 25 kda þ 35 kda proteinsð Þ½ Â 100: Aminoglycoside treatment Aminoglycoside treatment of homozygous Cftr-/- mice carrying the FABP-hCFTR-G542X transgene was initiated 16 days after birth (1 week before weaning). Login to comment 91 ABCC7 p.Gly542* Results Amikacin suppresses the hCFTR-G542X premature stop mutation in a mammalian in vitro translation system The ability of an aminoglycoside to suppress a premature stop mutation depends on several factors, including the specific features of its chemical structure, the identity of the stop codon to be suppressed, and the sequence context surrounding the stop codon in the mRNA [4, 5]. Login to comment 93 ABCC7 p.Gly542* Although amikacin has been shown to suppress premature stop mutations in several mammalian mRNAs in vitro [5, 17], it has not yet been shown to suppress the CFTR-G542X mutation in vitro. Login to comment 94 ABCC7 p.Gly542* We previously reported that gentamicin and tobramycin suppressed the hCFTR-G542X mutation in a mammalian RRL translation system [20]. Login to comment 96 ABCC7 p.Gly542* Accordingly, we first asked whether amikacin, an aminoglycoside commonly used to treat bacterial infections, could also suppress the hCFTR-G542X mutation using the RRL translation system. Login to comment 97 ABCC7 p.Gly542* Initially, we cloned the hCFTR-G542X premature stop codon and six flanking codons on either side into a readthrough reporter plasmid (Fig. 1) [20]. Login to comment 98 ABCC7 p.Gly542* ABCC7 p.Gly542* In this reporter system, a 25-kDa protein is produced if termination occurs at the G542X premature stop mutation, while a 35-kDa protein is produced if the G542X stop codon is suppressed. Login to comment 99 ABCC7 p.Gly542* This construct was expressed in a RRL-based coupled transcription/translation system in the presence of increasing concentrations of gentamicin or amikacin, and the concentration of each aminoglycoside that induced the maximum level of readthrough of the G542X mutation (without inhibiting total protein synthesis) was determined. Login to comment 100 ABCC7 p.Gly542* While both compounds were found to suppress termination at the G542X mutation, gentamicin induced readthrough at much lower concentrations than amikacin. Login to comment 101 ABCC7 p.Gly542* The maximum level of G542X readthrough induced by amikacin (11%) was similar to that induced by gentamicin (14%). Login to comment 106 ABCC7 p.Gly542* These results demonstrate that amikacin can effectively suppress the hCFTR-G542X mutation without inhibiting total protein synthesis as observed with gentamicin. Login to comment 112 ABCC7 p.Gly542* ABCC7 p.Gly542* In a previous study, we reported that the subcutaneous injection of 34 mg/kg gentamicin once daily suppressed the G542X mutation in Cftr-/- hCFTR-G542X mice and restored a significant level of functional hCFTR protein [20]. Login to comment 114 ABCC7 p.Gly542* In the current study, we sought to determine whether aminoglycosides could suppress the hCFTR-G542X mutation in our mouse model when administered at concentrations that produced serum levels well below the maximum recommended for humans. To determine an acceptable dose of each aminoglycoside, mice were injected subcutaneously with various concentrations of each compound. Login to comment 118 ABCC7 p.Gly542* The hCFTR-G542X UGA mutation (boxed) and surrounding context are indicated by the expanded sequence. Login to comment 119 ABCC7 p.Gly542* Termination at the G542X mutation results in a 25-kDa protein, while suppression of that mutation allows continued elongation and the production of a 35-kDa protein. Login to comment 120 ABCC7 p.Gly542* b Data showing suppression of the G542X mutation in an RRL in vitro translation system. Login to comment 121 ABCC7 p.Gly542* c Quantitation of G542X readthrough from data shown in b samples were then collected at specified times after administration and serum concentrations were determined using FPIA (Fig. 2). Login to comment 129 ABCC7 p.Gly542* ABCC7 p.Gly542* A clinical dose of gentamicin suppresses the hCFTR-G542X mutation but restores only a barely detectable amount of hCFTR protein We initially used an immunofluorescence assay to ask whether a once daily, low dose of 5 mg/kg gentamicin can suppress the hCFTR-G542X mutation. Login to comment 131 ABCC7 p.Gly542* A similar immunofluorescence assay was performed using intestinal tissues from untreated Cftr-/- hCFTR-G542X mice or from mice treated with either 34 mg/kg or 5 mg/kg gentamicin (Fig. 3a). Login to comment 133 ABCC7 p.Gly542* In the samples treated with the hCFTR-specific antiserum, no hCFTR protein was detected in the intestinal tissue from untreated Cftr-/- hCFTR-G542X mice, while a strong hCFTR protein signal was detected at the apical surface of epithelial cells in the submucosal glands of the duodenum from mice treated with 34 mg/kg gentamicin. Login to comment 135 ABCC7 p.Gly542* ABCC7 p.Gly542* These results suggest that this lower, clinically relevant dose of gentamicin can suppress the G542X mutation and restore hCFTR protein in Cftr-/- hCFTR-G542X mice, although the level of hCFTR protein detected was substantially less than was observed in animals treated with the higher dose. Login to comment 136 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* A clinical dose of amikacin suppresses the hCFTR-G542X mutation and restores a significant amount of hCFTR protein expression We next asked whether amikacin could suppress the hCFTR-G542X mutation in the Cftr-/- hCFTR-G542X mouse. Login to comment 138 ABCC7 p.Gly542* Because 34 mg/kg gentamicin was well-tolerated by Cftr-/- hCFTR-G542X mice in our previous study [20] and the doses of amikacin routinely recommended for human use are several folds higher than gentamicin [28], we initially chose to compare a high dose of 170 mg/kg amikacin and a low dose of 15 mg/kg amikacin. Login to comment 140 ABCC7 p.Gly542* To determine the ability of amikacin to suppress the hCFTR-G542X mutation, the same administration protocol was used and intestinal tissues from mice were assayed by immunofluorescence using either preimmune serum or an hCFTR-specific polyclonal antiserum. Login to comment 141 ABCC7 p.Gly542* No hCFTR protein was detected in samples from untreated Cftr-/- hCFTR-G542X mice using hCFTR-specific antiserum or in samples from treated mice using preimmune serum. Login to comment 144 ABCC7 p.Gly542* These results indicate that amikacin can restore a significant amount of hCFTR protein in the Cftr-/- hCFTR-G542X mice when administered at either a very high dose or at a lower dose that produces a peak serum level well within the recommended clinical range. Login to comment 147 ABCC7 p.Gly542* The aminoglycoside concentration in each serum sample was determined by fluorescence polarization immunoassay Analysis of hCFTR activity in Cftr-/- hCFTR-G542X mice after treatment with clinical doses of gentamicin or amikacin The hCFTR protein is a cyclic adenosine monophosphate (cAMP)-activated chloride channel that facilitates transepithelial chloride conductance upon activation by cAMP agonists such as forskolin. Login to comment 148 ABCC7 p.Gly542* We previously demonstrated that cAMP-dependent transepithelial chloride conductance could be detected in intestinal tissues of Cftr-/- hCFTR-G542X mice that had been treated with 34 mg/kg gentamicin [20]. Login to comment 150 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Figure 4 shows representative short-circuit current tracings obtained from Cftr-/- hCFTR-G542X mouse intestinal tissues that were harvested from untreated Cftr-/- hCFTR-G542X mice, or from Cftr-/- hCFTR-G542X mice treated once daily with either 5 mg/kg gentamicin or 15 mg/kg amikacin for 2 to 3 weeks. Login to comment 152 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Table 1 provides a summary of data collected from short-circuit current tracings of intestinal tissues harvested from untreated Cftr+/+ hCFTR-G542X mice, +/+ hCFTR-G542X mice treated with low doses of gentamicin or amikacin, untreated Cftr-/- hCFTR-G542X mice, and Cftr-/- hCFTR-G542X mice treated with high or low doses of gentamicin or amikacin. Login to comment 158 ABCC7 p.Gly542* a Samples from the duodenum of homozygous Cftr -/-hCFTR-G542X mice (untreated or treated with 5 or 34 mg/kg gentamicin). Login to comment 159 ABCC7 p.Gly542* b Samples from the duodenum of homozygous Cftr-/-hCFTR-G542X mice (untreated or treated with 15 or 170 mg/kg amikacin). Login to comment 163 ABCC7 p.Gly542* A P<0.05 value was considered as significant In untreated Cftr-/- hCFTR-G542X mice used as negative controls, we observed a cAMP-stimulated short-circuit current in only 8% of the samples (1/12), resulting in an average current of only 0.2 μA/cm2 . Login to comment 164 ABCC7 p.Gly542* In our experimental samples, we found that 63% of samples (5/8) from Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin yielded cAMP-activated short-circuit currents after forskolin addition, resulting in an average current of 1.67 μA/cm2 . Login to comment 165 ABCC7 p.Gly542* In contrast, 35% of samples (7/20) from Cftr-/- hCFTR-G542X mice treated with 5 mg/kg of gentamicin yielded cAMP-stimulated short-circuit currents, resulting in an average current of 0.82 μA/cm2 . Login to comment 167 ABCC7 p.Gly542* They also demonstrate that the administration of a clinically relevant dose of gentamicin to Cftr-/- hCFTR-G542X mice can restore a statistically significant increase (P value<0.05) in cAMP-stimulated chloride currents relative to untreated controls, consistent with a partial restoration of CFTR protein and activity. Login to comment 169 ABCC7 p.Gly542* We found that treatment of Cftr-/- hCFTR-G542X mice with 170 mg/kg of amikacin once daily for 23 weeks yielded a short-circuit response in 75% of intestinal tissues assayed (9/12), resulting in an average current of 1.77 μA/cm2 . Login to comment 173 ABCC7 p.Gly542* Discussion The results of this study show that amikacin, like gentamicin, can effectively suppress the hCFTR-G542X stop mutation in a transgenic CF mouse model. Login to comment 174 ABCC7 p.Gly542* In fact, we found that the low dose of amikacin tested in this study appears to suppress the hCFTR-G542X mutation more effectively than the low dose of gentamicin. Login to comment 182 ABCC7 p.Gly542* a Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse without any prior aminoglycoside treatment. Login to comment 183 ABCC7 p.Gly542* b Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse after 5 mg/kg gentamicin treatment once daily for 2-3 weeks. Login to comment 184 ABCC7 p.Gly542* c Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse after 15 mg/kg amikacin treatment once daily for 2-3 weeks. Login to comment 210 ABCC7 p.Gly542* The expression of the human CFTR-G542X cDNA in this animal will undoubtedly be different than the expression of endogenous mouse Cftr that contains a premature stop mutation. Login to comment 212 ABCC7 p.Gly542* In our study, we found that the administration of amikacin at doses that produced serum levels within the clinically accepted range could suppress the hCFTR-G542X mutation and restore functional hCFTR protein. Login to comment 213 ABCC7 p.Gly542* While gentamicin was also found to suppress the hCFTR-G542X mutation when administered in doses that produced serum levels in the accepted clinical range, the levels of suppression appeared to be significantly less than those observed with amikacin. Login to comment