Evaluating the efficiency and specificity of stop codon suppression therapy

描述（由申请人提供）：终止密码子抑制疗法是一种基于核糖体结合药物降低过早终止密码子突变时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读取部分恢复全长蛋白的表达，并且在某些情况下可以改善疾病症状。由于帧内过早终止密码子占所有已知致病突变的约15%，因此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。众所周知的氨基糖苷庆大霉素或新发现的停止密码子抑制化合物阿特鲁伦的临床试验令人鼓舞，尽管在一些患者中缺乏反应，并且只是部分恢复蛋白质表达，这迫使人们额外努力寻找具有更高活性、特异性和更少副作用的新药。尽管在开发这些疗法方面做出了广泛的努力，但关键的障碍仍然存在，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。本研究的目标分为两部分：1)开发一种定量方法，以确定停止密码子抑制处理对体内和细胞培养中转录组翻译和mRNA丰度的影响；2)开发一种定量分析方法，作为评估新候选化合物的效率和特异性的平台。我们将测试这一假设，即一些天然含有“过早”终止密码子的生理性mrna可能容易受到终止密码子抑制。这些包括具有调控短5'UTR开放阅读框或长3' utr的细胞转录本，硒蛋白mrna，以及由转座元件或无功能假基因编码的转录本。这些信息上诱导的停止密码子通读可能对mRNA稳定性有进一步的影响，因为无义介导的衰变途径对调控的易感性发生了改变。该方法利用了核糖体分析的最新发展；一种基于深度测序的方法，以核苷酸精度量化核糖体密度和数千种mrna的定位。目的1核糖体分析和RNA-Seq方法将被开发，以评估杜氏肌营养不良小鼠遗传（停止密码子突变）模型中停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停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面影响，并开发适当的方法来评估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，并有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.