Data from: PTC124 Targets Genetic Disorders Caused by Nonsense Mutations
UMass Chan AffiliationsDepartment of Microbiology and Physiological Systems
KeywordsBiochemistry, Biophysics, and Structural Biology
Cellular and Molecular Physiology
Chemicals and Drugs
Congenital, Hereditary, and Neonatal Diseases and Abnormalities
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AbstractManuscript abstract: Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from less than 1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/36443
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Methodology for RNA analysis: Quantitative real-time PCR was performed using primers for LUC and dystrophin mRNAs, with the levels of 18S rRNA and GAPDH mRNA serving as normalization factors for the amount of starting material. Northern blot analysis was performed using probes specific to LUC mRNA, with the level of GAPDH mRNA serving as a control for loading. High density oligonucleotide arrays (Affymetrix U133+, 2.0), containing 54,675 probe sets/chip) were used to analyse expression profiles of PTC124-treated, gentamicin-treated and untreated HEK293 cells. cRNA sample preparation, array hybridization and scanning followed protocols recommended by the manufacturer (http://www.affymetrix.com/estore/). The hybridization intensity (signal) of each transcript was determined using the Affymetrix Microarray Suite 5.0 software package. Intensity values were scaled such that the overall fluorescence intensity of each array was equivalent. Scaling factors for each experimental set varied by less than 20%. Transcript levels specifically enhanced or reduced by drug treatment were identified by applying several stringent standards to the raw data, including: (a) the signal values of a transcript in the treated and untreated samples had to have a minimal level of at least 26 units and a relative change of at least twofold; (b) these changes had to be reproducible in five of the six independent replicate experiments; and (c) these changes had to demonstrate statistically significant P-values ≤ 0.05 in a group t-test. The GeneSpring (version 7; http://www.silicongenetics.com) and GENECLUSTER (version 1.0; http://www-genome.wi.mit.edu) software packages were used to identify transcripts that were differentially expressed.
This dataset (.xls file, 24 MB) is the primary RNA analysis data for the following published study: Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, Sweeney HL. PTC124 targets genetic disorders caused by nonsense mutations. Nature. 2007 May 3;447(7140):87-91. Epub 2007 Apr 22. doi:10.1038/nature05756. PubMed PMID: 17450125.