Activating Protein Expression Using Antisense Oligonucleotides

使用反义寡核苷酸激活蛋白质表达

• 批准号: 10350613
• 负责人: Zachary Kartje
• 金额: $ 6.98万
• 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/10350613
• 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.

项目概要 二倍体生物体中单倍体不足的特征是基因的工作副本和非功能副本， 导致基因产物（即蛋白质）数量不足，这会破坏正常的细胞功能。 导致多种疾病的有效基因激活策略尚未出现。 之所以被确定是因为抑制蛋白质产生的机制尚不清楚。 反义寡核苷酸 (ASO) 是小型、可预测和可编程的工具，可以通过化学方法 设计用于直接控制 RNA 的稳定性、加工和翻译，使它们可用于 先前对野生型细胞的蛋白质生产机制的剖析表明了空间阻断。 ASO 可以阻断核糖体的替代翻译起始位点并直接剪接因子以增加蛋白质 此外，ASO“缺口聚体”包含两侧经过化学修饰的 DNA 中心区域。 核苷酸，可以降解负向调节蛋白质表达的 RNA（例如反义转录物）。 这些策略在单倍体不足的情况下的有效性尚未得到研究。 在 Jonathan Watts 博士（ASO 合成和化学）和合作者：Athma Pai 博士的指导下 （RNA 处理和生物信息学）、Anastasia Khvorova 博士（ASO 递送和神经生物学）和 Xandra Breakefield（肿瘤抑制综合征），该提案旨在设计和应用化学- 改进的 ASO 系统地研究内源性蛋白质抑制机制并确定关键 调节全长蛋白质翻译的因子，使用 NF1 基因作为肿瘤抑制因子。 抑制 Ras/MAPK 信号传导的 NF1 单倍体不足会导致 1 型神经纤维瘤病，这是一种遗传性疾病。 NF1 基因座的特点是不受控制的神经细胞增殖和其他并发症。 本研究的模型，因为它拥有两个替代翻译起始位点——上游开放阅读 成熟 mRNA 5' 非翻译区 (UTR) 中的框架 (uORF) 被多个反义重叠； 转录本；并且可能会发生结合 NF1 5'UTR uORF 的非生产性剪接。 目标 1 将测试这些 ASO 线索的功效以启动。 目标 2 将设计并应用 ASO gapmer。 靶向并降解 NF1 反义转录物并确定其对 NF1 蛋白表达的影响 Aim 3。 将分离 NF1 新生 RNA 并对其进行测序，以识别隐藏的剪接位点，然后设计 ASO 来阻断。 为了实现所有目标，候选 ASO 将被转染到这些位点并提高前 mRNA 剪接效率。 SH-SY5Y 神经母细胞瘤细胞（表达 NF1）将用于批量筛选。 在野生型和 NF1+/- 单倍体不足的神经元和雪旺细胞中进行了优化和测试。 将通过测量 Ras/MAPK 激活来评估激活的 NF1 蛋白。 了解蛋白质表达如何调节，并可能为纠正单倍体不足的策略提供信息。