Stop codon suppression therapy is a treatment strategy based on the ability of ribosome binding pharmaceutical agents to reduce the fidelity of translation termination at premature stop codon mutations. The resulting drug-induced read-through of premature stop codon mutations partially restores expression of full- length protein and can in some cases ameliorate disease symptoms. As in-frame premature stop codons account for ~15% of all known disease causing mutations, stop codon suppression therapy has the potential to treat hundreds of thousands of patients with a variety of genetic disorders. Clinical trials testing the well known aminoglycoside gentamicin or the newly identified stop codon suppression compound Ataluren have been encouraging, although the lack of response in some patients and only partial restoration of protein expression has compelled additional efforts to identify new drugs with increased activity, specificity, and reduced side- effects. Despite extensive efforts to develop these therapies, critical barriers remain, including lack of appropriate methodologies to examine the primary and secondary effects of candidate stop codon suppression drugs on translation. The goals of this proposal are two part: 1) to develop a quantitative methodology to identify the effects of stop codon suppression treatments on translation and mRNA abundance across the transcriptome both in vivo and in cell culture, and 2) to develop a quantitative assay that can be used as a platform to evaluate the efficiency and specificity of new candidate compounds. We will test the hypothesis that some physiological mRNAs that naturally contain 'premature'stop codons are likely susceptible to stop codon suppression. These include cellular transcripts with regulatory short 5'UTR open reading frames or long 3'UTRs, selenoprotein mRNAs, and transcripts encoded by transposable elements or non-functional pseudogenes. Induced stop codon read-through on these messages may have further consequences for mRNA stability due to altered susceptibility to regulation by the nonsense mediated decay pathway. The approach capitalizes on the recent development of ribosome profiling;a deep-sequencing based methodology that quantifies ribosome density and localization on thousands of mRNAs with nucleotide precision.
In aim 1 ribosome profiling and RNA-Seq methods will be developed to evaluate changes in translation and mRNA abundance arising from stop codon suppression therapies in a mouse genetic (stop codon mutation) model of Duchenne Muscular Dystrophy.
In aim 2 we will apply this methodology to primary cultured cells treated with stop codon suppression drugs to demonstrate the feasibility of screening new compounds in patient cells. A deeper understanding of the full range of drug-induced effects on protein expression and the development of appropriate methodologies to evaluate these effects for each compound under consideration will inform the design and interpretation of clinical trials and potentially lead to the development of more effective therapeutic strategies.
Stop codon suppression therapy is a rapidly developing approach to personalized medicine designed to treat a class of mutations, premature stop codons, rather than a particular disease. As premature stop codon mutations account for ~15% of all known disease causing mutations, this therapeutic approach has broad implications for a large number of patients and many different genetic disorders. The goals of this proposal are two part: 1) to identify the effects of stop codon suppression treatments on translation and mRNA abundance across the transcriptome both in tissues and in cultured cells, and 2) to develop a quantitative assay to evaluate the efficiency and specificity of new candidate stop codon suppression drugs.
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