Evaluating the efficiency and specificity of stop codon suppression therapy

描述(申请人提供)：终止密码子抑制疗法是一种基于核糖体结合药物在过早终止密码子突变时降低翻译终止保真度的治疗策略。由此产生的药物诱导的过早终止密码子突变的通读部分恢复了全长蛋白质的表达，在某些情况下可以改善疾病症状。由于框内过早终止密码子约占所有已知疾病引起的突变的15%，终止密码子抑制疗法有可能治疗数十万患有各种遗传疾病的患者。测试著名的氨基糖苷类庆大霉素或新发现的终止密码子抑制化合物Ataluren的临床试验令人鼓舞，尽管一些患者缺乏反应，仅部分恢复了蛋白质表达，这迫使人们做出额外努力，寻找活性更高、特异性更强、副作用更少的新药。尽管为开发这些疗法做出了广泛的努力，但仍然存在严重的障碍，包括缺乏适当的方法来检查候选终止密码子抑制药物对翻译的主要和次要影响。这项建议的目标有两个部分：1)开发一种定量方法来确定终止密码子抑制处理对体内和细胞培养中转录组的翻译和mRNA丰度的影响，以及2)开发一种可以作为评估新候选化合物的有效性和特异性的平台的定量分析方法。我们将测试这一假设，即一些自然包含“过早”终止密码子的生理性mRNAs可能容易停止密码子抑制。这些基因包括具有调控短的5‘非编码区开放阅读框架或长的3’非编码区的细胞转录本，硒蛋白mRNAs，以及由转座元件或非功能假基因编码的转录本。这些信息上的诱导终止密码子通读可能会对mRNA的稳定性产生进一步的影响，这是因为改变了对无义介导的衰变途径调节的敏感性。该方法利用了核糖体图谱的最新发展；这是一种基于深度测序的方法，可以精确地量化核糖体密度和在数千个mRNA上的定位。在目标1中，将开发核糖体图谱和RNA-Seq方法来评估杜氏肌营养不良症小鼠遗传模型(终止密码子突变)中终止密码子抑制治疗引起的翻译和mRNA丰度的变化。在目标2中，我们将把这种方法应用于用终止密码子抑制药物处理的原代培养细胞，以证明在患者细胞中筛选新化合物的可行性。更深入地了解药物对蛋白质表达的全方位影响，并开发适当的方法来评估所考虑的每种化合物的这些影响，将为临床试验的设计和解释提供信息，并可能导致开发更有效的治疗策略。

DESCRIPTION (provided by applicant): 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.