Readthrough of dystrophin stop codon mutations induced by aminoglycosides

Howard, Michael; Anderson, Christine B.; Fass, U.; Khatri, Shikha; Gesteland, Raymond F.; Atkins, John F.; Flanigan, Kevin M.

doi:10.1002/ana.20052

Cited by 104 publications

(79 citation statements)

References 14 publications

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“…This is being critically evaluated for cystic fibrosis (13)(14)(15)(16) and muscular dystrophy (17)(18)(19)(20)(21)(22). After animal studies, gentamicin-induced CFTR protein was recently reported to improve transmembrane conductance across the nasal mucosa in a group of 10 patients with homozygous PTC mutations in the CTRF gene; patients with the more common del508F mutation were used as controls and showed no improvement of conductance (16).…”

Section: Discussionmentioning

confidence: 99%

“…Studies treating mdx mice with gentamicin were reported to restore dystrophin function in skeletal muscle (17). However, the muscular dystrophy clinical trials have not been encouraging and a recent report failed to confirm the earlier mouse experiments (18,20,21). Aminoglycoside-induced readthrough levels have varied from 2% to 10% (21).…”

Section: Discussionmentioning

confidence: 99%

“…However, the muscular dystrophy clinical trials have not been encouraging and a recent report failed to confirm the earlier mouse experiments (18,20,21). Aminoglycoside-induced readthrough levels have varied from 2% to 10% (21). Aminoglycoside-induced PTC read-through has also been documented in other disorders, such as Hurler's (23), diabetes insipidus (24,25), cystinosis (26), and X-linked retinitis pigmentosa (27).…”

Section: Discussionmentioning

confidence: 99%

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Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

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Cells were irradiated with 10 Gy and were incubated for 45 min at 37°C before fixation on coverslips. ATM pS1981 IRIFs were not observed in untreated cells; maximum intensity of staining was seen in the nuclei of cells exposed to 75 g͞ml. Nonirradiated cells, untreated or exposed to comparable concentrations of geneticin, showed only occasional foci. A taxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with onset in early childhood, resulting from mutations in the A-T mutated (ATM) gene (1). The ATM protein is a hierarchical serine-threonine kinase, phosphorylating many substrates involved in repair of doublestranded DNA breaks, control of cell cycle checkpoints, and responses to oxidative stress, as well as in radiosensitivity, cancer susceptibility, immune function, and neurological development (2). ATM protein levels are undetectable in the cells of most A-T patients by conventional testing. However a few patients, often with a milder phenotype, have some detectable ATM protein (3,4). This finding encouraged us to seek compounds, such as aminoglycosides, that have the potential to read through premature termination codons and restore ATM protein function (5, 6).To test these effects, we selected 13 lymphoblastoid cell lines (LCLs) with primary premature termination codon (PTC) mutations from a repository of Ͼ400 lines derived from A-T patients. Herein, we show representative data from 5 of the 13 LCLs. Using protein truncation testing (PTT) driven by plasmid templates containing PTC mutations in the ATM gene (7), we observed in vitro read-through effects of various magnitudes with three of four aminoglycosides tested. Full-length ATM protein was also documented ex vivo by immunoprecipitation. Correction of radioresistant DNA synthesis and radiosensitivity, as well as autophosphorylation of ATM, suggested that this readthrough produces functional ATM protein. Experimental ProceduresCell Lines. Lymphoblastoid cell lines were maintained in RPMI medium 1640 (Invitrogen) with 15% FBS (HyClone) and 1% penicillin͞streptomycin (Invitrogen) at 37°C and 5% CO 2 . In ex vivo experiments, aminoglycosides were added daily into culture media at the indicated doses and times. Because streptomycin is also an aminoglycoside that can perturb proofreading by binding to another ribosomal site, ex vivo experiments were performed with or without streptomycin; no differences were noted.Mutations. We selected only PTC mutations that resulted directly from the mutation, i.e., primary PTC mutations, and not those caused indirectly by upstream mutations or aberrant splicing, because the latter would have no potential therapeutic benefits. The LCLs carried the following mutations (www.benaroyaresearch.org͞bri investigators͞atm.htm): AT185LA, homozygous 3673C 3 T (a UAA G stop codon in PTT fragment 4); TAT51, homozygous 5623C 3 T (a UGA C stop codon in PTT fragment 5); AT187LA, homozygous 5908C 3 T (a UAA G stop codon in PTT fragment 6); AT153LA, homozygous 8977C 3 T (a UGA A stop codon in fragment 8); and AT...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

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“…Dual luciferase assays were performed using a dual luciferase assay kit (Promega Corp.), and light emission was quantitated using a Berthold Lumat LB9507 luminometer. The dual luciferase reporters used to monitor readthrough of stop codons in mammalian cells have been described previously (17,18). The percentage of readthrough was defined as the firefly/Renilla luciferase activity (nonsense) divided by firefly/Renilla luciferase activity (sense) ϫ 100.…”

Section: Methodsmentioning

confidence: 99%

“…To examine this question, we used a dual luciferase readthrough reporter system in cultured HEK293T cells. In this system, the Renilla and firefly luciferase reporter genes are located upstream and downstream of an in-frame UGA stop codon, respectively (17,18) (Fig. 1A).…”

Section: Treatment Of Cultured Cells With Paa and Gentamicinmentioning

confidence: 99%

Poly-l-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model

Keeling

Fan

et al. 2009

Journal of Biological Chemistry

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Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-L-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20 -40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr ؊/؊ hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach.Previous studies have shown that aminoglycosides such as gentamicin and amikacin can suppress translation termination at disease-causing premature stop (nonsense) mutations and partially restore the expression of functional proteins in mammalian cells (for a review, see Ref. 1). In particular, gentamicin has been shown to suppress nonsense mutations and partially restore protein expression in mouse models of Duchenne muscular dystrophy (2) and cystic fibrosis (CF) 2 (3, 4). However, the use of aminoglycosides is commonly associated with serious side effects, including nephrotoxicity and ototoxicity (5, 6). The high dose of gentamicin (34 mg/kg) initially used to demonstrate readthrough in mouse models also resulted in serum concentrations that were far in excess of their maximum clinically recommended levels (2-4). More recently, we demonstrated that a lower dose of gentamicin (5 mg/kg) produced peak serum concentrations in a CF mouse model that were within the accepted clinical range for these compounds (4). Functional CFTR protein was produced under those conditions, as shown by immunofluorescence and short circuit current measurements. However, the level of suppression obtained was significantly less than was observed with higher doses. Consistent with the efficacy of these clinically relevant doses in mice, small clinical trials have suggested that g...

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Aminoglycosides and other nonsense suppression therapies for the treatment of dystrophinopathy

Sampson

Vardeny²,

Flanigan

2009

Cochrane Database of Systematic Reviews

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Readthrough of dystrophin stop codon mutations induced by aminoglycosides

Cited by 104 publications

References 14 publications

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Poly-l-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model

Aminoglycosides and other nonsense suppression therapies for the treatment of dystrophinopathy

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