Activating Protein Expression Using Antisense Oligonucleotides

使用反义寡核苷酸激活蛋白质表达

• 批准号: 10156330
• 负责人: Zachary Kartje
• 金额: $ 6.64万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10156330
• 关键词: 5' Untranslated Regions; Algorithms; Alleles; Antisense Oligonucleotides; Area; Binding; Binding Sites; Bioinformatics; Cell Proliferation; Cell physiology; Cells; Chemical Engineering; Chemicals; Chemistry; DNA; Defect; Diploidy; Disease; Engineering; Ensure; Exons; Future; Gene Activation; Gene Expression; Genes; Genetic Diseases; Genetic Transcription; Health; Intervention; Label; Length; MAP Kinase Gene; Measures; Mentorship; Messenger RNA; Modeling; Mutate; NF1 gene; Neurobiology; Neurofibromatosis 1; Neurons; Normal Cell; Nucleic Acids; Nucleotides; Open Reading Frames; Organism; Pathway interactions; Patients; Peripheral Nerve Sheath Neoplasm; Phenotype; Process; Production; Proteins; RNA; RNA Processing; RNA Splice Sites; RNA Splicing; Repression; Reverse Transcriptase Polymerase Chain Reaction; Ribosomes; Schwann Cells; Signal Transduction; Site; Study models; Syndrome; Synthesis Chemistry; Technology; Testing; Thiouridine; Transcript; Translation Initiation; Translations; Tumor Suppressor Proteins; Work; base; cell type; design; efficacy testing; gene product; improved; knock-down; lead optimization; mRNA Precursor; nervous system disorder; neuroblastoma cell; novel; programs; protein expression; screening; therapeutic development; tool; transcriptome sequencing; translation factor

PROJECT SUMMARY Haploinsufficiency in diploid organisms is characterized by a working copy and nonfunctional copy of a gene, resulting in an insufficient amount of gene product (i.e., protein). This disrupts normal cell function, and can cause a myriad of diseases. Effective gene activation strategies for correcting haploinsufficiency have not been identified because the mechanisms that repress protein production are unclear. Antisense oligonucleotides (ASOs) are small, predictable, and programmable tools that can be chemically engineered to directly control the stability, processing, and translation of RNA, making them useful for dissecting mechanisms of protein production. Previous work in wild-type cells demonstrates that steric blocking ASOs can block alternative translation start sites from ribosomes and direct splicing factors to increase protein levels. Moreover, ASO “gapmers”, which contain a central region of DNA flanked by chemically-modified nucleotides, can degrade RNAs that negatively regulate protein expression (e.g. antisense transcripts). Yet, the efficacy of these strategies in a haploinsufficiency context has not been investigated. With guidance from Dr. Jonathan Watts (ASO synthesis and chemistry), and collaborators: Dr. Athma Pai (RNA processing and bioinformatics), Dr. Anastasia Khvorova (ASO delivery and neurobiology), and Dr. Xandra Breakefield (tumor-suppressor syndromes), this proposal seeks to design and apply chemically- modified ASOs to systematically investigate endogenous protein repression mechanisms and identify key factors modulating full-length protein translation, using the NF1 gene as a model. NF1 is a tumor suppressor that inhibits Ras/MAPK signaling. NF1 haploinsufficiency causes neurofibromatosis type 1, a genetic disorder characterized by uncontrolled nerve cell proliferation and other complications. The NF1 locus is an excellent model for this study because it possesses two alternative translation start sites – upstream open reading frames (uORFs) in the 5’ untranslated region (UTR) of the mature mRNA; is overlapped by several antisense transcripts; and likely undergoes unproductive splicing. Steric blocking ASOs that bind NF1 5’UTR uORFs have been synthesized and promising leads identified. Aim 1 will test the efficacy of these ASO leads to initiate translation at the primary start site and increase protein expression. Aim 2 will design and apply ASO gapmers to target and degrade NF1 antisense transcripts and determine their effect on NF1 protein expression. Aim 3 will isolate and sequence NF1 nascent RNA to identify cryptic splice sites. ASOs will then be designed to block these sites and improve pre-mRNA splicing efficiency. For all aims, candidate ASOs will be transfected into SH-SY5Y neuroblastoma cells (which express NF1) for bulk screening. Successful candidates will then be tested and optimized in wild-type and NF1+/- haploinsufficent neurons and Schwann cells. Functionality of activated NF1 protein will be assessed by measuring Ras/MAPK activation. This project will increase our understanding of how protein expression is regulated, and may inform strategies to correct haploinsufficiency.

项目摘要 二倍体生物中的单倍不适的特征是基因的工作副本和非功能副本， 导致基因产物不足（即蛋白质）。这会破坏正常的细胞功能，并且可以 引起无数疾病。有效纠正单倍不足的基因激活策略尚未 被确定是因为反映蛋白质产生的机制尚不清楚。 反义寡核苷酸（ASO）很小，可预测且可编程工具，可以化学上可以化学 设计为直接控制RNA的稳定性，处理和翻译，使其可用于 解剖蛋白质产生的机制。野生型细胞中的先前工作表明，空间阻塞 ASO可以阻止核糖体和直接剪接因子的替代翻译起始位点以增加蛋白质 水平。此外，ASO“ Gapmers”，其中包含一个由化学修饰的DNA的中心区域 核苷酸会降解负调节蛋白质表达的RNA（例如反义转录本）。然而， 尚未研究这些策略在单倍不足的情况下的效率。 在Jonathan Watts博士（ASO合成和化学）和合作者的指导下：Athma Pai博士 （RNA处理和生物信息学），Anastasia Khvorova博士（ASO递送和神经生物学）和博士 Xandra Breakefield（肿瘤抑制剂综合征），该提案旨在设计和应用化学应用 修改的ASO系统地研究内源性蛋白表达机制并识别钥匙 使用NF1基因作为模型调节全长蛋白质翻译的因素。 NF1是肿瘤抑制剂 这抑制了RAS/MAPK信号。 NF1单倍不足导致1型神经纤维瘤病，一种遗传疾病 以不受控制的神经细胞增殖和其他并发症为特征。 NF1基因座很棒 这项研究的模型是因为它具有两个替代的翻译起始站点 - 上游开放阅读 成熟mRNA的5'未翻译区（UTR）中的框架（UORF）；被几种反义重叠 成绩单；并且可能经历了无生产的剪接。绑定NF1 5'Utr uorfs的空间阻止ASO 已经合成并确定了潜在客户。 AIM 1将测试这些ASO的效率，以启动 在初级开始位点的翻译并增加蛋白质表达。 AIM 2将设计并应用ASO Gapmers 靶向和降解NF1反义转录本，并确定其对NF1蛋白表达的影响。目标3 将分离和序列NF1新生RNA以识别加密剪接位点。然后将设计ASOS以阻止 这些位点并提高mRNA剪接效率。对于所有目标，候选人ASO将被翻译成 SH-SY5Y神经母细胞瘤细胞（表达NF1）用于大量筛选。成功的候选人将是 在野生型和NF1 +/-单倍体神经元和雪旺氏细胞中进行了测试和优化。功能 活化的NF1蛋白将通过测量RAS/MAPK激活来评估。这个项目将增加我们的 了解如何调节蛋白质表达，并可能告知策略以纠正单倍度不足。