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

Abstract

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...

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

“…In our study, we focused on nonsense or frameshift mutations leading to PTC resulting in an almost complete absence of particular key proteins involved in lysosomal function and cell homeostasis maintenance. Preclinical studies with gentamicin or PTC124 had succeeded by enhancing stop codon readthrough and were able to be used as therapeutic agents for genetic diseases [9][10][11][12][13][14][15][16][17][18][36][37][38][39][40][41][42]. PTC124 has been successfully extended into clinical trials [18,43], although preliminary results are not clear for Duchenne muscular dystrophy [44].…”

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

“…Taken together, these findings suggest that therapeutic benefits in A-T patients might be achieved if even modest increases in functional ATM protein levels could be achieved. In previous studies we were able to induce ATM protein levels in LCLs from A-T patients with nonsense mutations by exposure to aminoglycosides that read through premature stop codons (22). Such mutations occur in Ϸ30% of A-T patients.…”

SMRT compounds abrogate cellular phenotypes of ataxia telangiectasia in neural derivatives of patient-specific hiPSCs

“…Mutation-targeted therapies, such as read-through compounds (RTCs) for nonsense mutations [10][11][12] and antisense morpholino oligonucleotides for splicing mutations [13][14][15] , have restored functional expression of the ATM protein in A-T patient-derived cells. Several leading RTCs from our previous studies induced expression of functional ATM protein in human nonsense mutation-containing lymphoblastoid cell lines (LCLs) and fibroblasts derived from A-T patients 11 .…”