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

鉴定新的人类靶点以抑制含有 PTC 的 mRNA 的衰变

• 批准号: 8995711
• 负责人: ANDREI ALEXANDROV
• 金额: $ 16.65万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8995711
• 关键词: Accounting; Animal Model; Ataxia Telangiectasia; Biochemical; Biological Assay; Cell Survival; Cell physiology; Cells; Complex; Cystic Fibrosis; Data; Disease; Dominant-Negative Mutation; Duchenne muscular dystrophy; Exons; Extravasation; Fluorescence; Generations; Genes; Genetic; Genetic Screening; Genetic study; Genomics; Health; Hereditary Disease; Human; Human Genetics; Institutes; Intervention; Investigational Therapies; Knock-out; Length; Libraries; Messenger RNA; Methods; Molecular; Mucopolysaccharidosis I H; Muscular Dystrophies; Mutation; Names; National Institute of Neurological Disorders and Stroke; Neurofibromatoses; Nonsense-Mediated Decay; Pathway interactions; Plasmids; Process; Proteins; Public Health; RNA Processing; Reading; Research Personnel; Retinoblastoma; Role; Severity of illness; Sorting - Cell Movement; System; Tay-Sachs Disease; Technology; Terminator Codon; Testing; Therapeutic; Toxic effect; Variant; Work; cell growth; empowered; forward genetics; gene product; genetic approach; genome-wide; in vivo; knock-down; knockout gene; new technology; novel; premature; research study; screening; targeted treatment; therapeutic target; tool; whole genome

 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.

 描述（由申请人提供）：NMD途径降解含有提前终止密码子（PTC）的mRNA。PTC突变占人类遗传疾病的三分之一，包括破坏性疾病的变体，如杜氏肌营养不良症，囊性纤维化和赫尔勒综合征; PTC通常是致癌的。目前，还没有治愈PTC起源的遗传性疾病。NMD途径代表PTC起源的遗传病症的潜在治疗靶标：已知其调节疾病严重程度; NMD的部分抑制可通过增加含PTC的mRNA的水平来辅助无义抑制疗法。由于NMD参与者本质上参与多种细胞过程，因此潜在的治疗性NMD抑制将受益于对NMD的人类特异性方面的理解。 然而，人类特异性NMD因子的范围仍然不确定，因为大多数NMD因子首先使用正向遗传学在模式生物中发现，然后才在人类中通过同源性发现。尽管人类NMD的机制与模型生物体中的机制显著不同（例如，在许多模型生物体中，外显子连接复合物对于NMD是不需要的），但由于该方法在人类细胞中的灵敏度/通量限制，从未使用无偏正向遗传学来询问其NMD途径。此外，必需多功能蛋白的传统敲除/敲低破坏了它们的所有功能，使NMD参与者的遗传鉴定复杂化/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，允许对人类细胞中的NMD因子进行超高通量正向遗传筛选;和（ii）对必需的多功能人类蛋白质的残基进行大规模体内询问以鉴定NMD所需的残基的方法。 该项目旨在使用这两种无偏倚的方法来（1）发现新的人类特异性NMD因子，为PTC起源的人类遗传性疾病提供新的干预靶点，以及（2）鉴定适合NMD抑制的多功能人类蛋白质残基簇，毒性有限，有助于PTC通读促进实验疗法。