Identification of New Human Targets to Inhibit Decay of PTC-containing mRNAs

 描述(申请人提供)：NMD途径降解含有提前终止密码子(PTC)的mRNAs。PTC突变占人类遗传病的三分之一，包括Duchenne肌营养不良症、囊性纤维化和Hurler综合征等破坏性疾病的变体；PTC通常是致癌的。目前，PTC起源的遗传性疾病还没有治愈方法。NMD通路代表了PTC起源的遗传性疾病的潜在治疗靶点：已知它可以调节疾病的严重程度；部分抑制NMD可以通过增加含有PTC的mRNAs的水平来辅助无意义抑制治疗。由于NMD玩家本质上涉及多个细胞过程，因此，了解NMD的人类特有方面将有助于潜在的治疗性NMD抑制。然而，人类特有的NMD因子的范围仍然不确定，因为大多数NMD因子是首先在模式生物中使用正向遗传学发现的，然后才在人类中通过同源性发现的。尽管人类NMD的机制与模式生物中的机制有很大的不同(例如，在许多模式生物中NMD不需要外显子连接复合体)，但由于这种方法在人类细胞中的敏感性/吞吐量限制，其NMD途径从未被无偏正向遗传学研究过。此外，传统的必需多功能蛋白质的敲除/敲除破坏了它们的所有功能，使NMD玩家的遗传鉴定复杂化/排除。为了使正向遗传学方法能够研究人类NMD，我开发了两种方法：(I)体内荧光扩增(Fireworks)方法，允许超高通量正向筛选人类细胞中的NMD因子；以及(Ii)一种在体内对必需的多功能人体蛋白残基进行大规模询问的方法，以确定NMD所需的残基。本项目的目的是使用这两种无偏见的方法来(1)发现新的人类特异性NMD因子，为PTC起源的人类遗传疾病提供新的干预靶点，(2)识别适合抑制NMD的毒性有限的多功能人类蛋白簇，以帮助促进PTC通读的实验治疗。

 DESCRIPTION (provided by applicant): The NMD pathway degrades mRNAs that contain a premature termination codon (PTC). PTC mutations account for one third of human genetic diseases including variants of devastating diseases like Duchenne muscular dystrophy, cystic fibrosis, and Hurler syndrome; PTCs are often carcinogenic. Currently, there is no cure for PTC-originating genetic disorders. NMD pathway represents a potential therapeutic target for PTC- originating genetic disorders: it is known to modulate disease severity; partial inhibition of NMD can assist nonsense suppression therapies by increasing the level of PTC-containing mRNAs. Since NMD players are intrinsically involved in multiple cellular processes, potential therapeutic NMD inhibition would therefore benefit from understanding of human-specific aspects of NMD. However, the scope of human-specific NMD factors remains undefined since the majority of NMD factors were first discovered in model organisms using forward genetics, and only then found by homology in human. Even though mechanisms of human NMD are significantly different from those in model organisms (e.g. the exon junction complex is not required for NMD in a number of model organisms), its NMD pathway has never been interrogated using unbiased forward genetics due to sensitivity/throughput limitations of this approach in human cells. Additionally, traditional knockouts/knockdowns of essential multi-functional proteins disrupt all their functions, complicating/precluding genetic identification of NMD players. To enable forward genetic approach to study human NMD, I developed two methods: (i) a method of in vivo fluorescence amplification (Fireworks) allowing ultra-high throughput forward genetic screening for NMD factors in human cells; and (ii) an approach to perform massive in vivo interrogation of residues of essential multi-functional human proteins to identify residues required for NMD. This project is aimed to use these two unbiased approaches to (1) discover new human-specific NMD factors to provide novel intervention targets for PTC-originating human genetic disorders, and (2) identify clusters of residues of multi-functional human proteins suitable for NMD inhibition with limited toxicity, aiding PTC readthrough-promoting experimental therapies.