Activating Protein Expression Using Antisense Oligonucleotides

项目摘要 二倍体生物中的单倍不足的特征在于基因的工作拷贝和非功能拷贝， 导致基因产物的量不足（即，蛋白质）。这会破坏正常的细胞功能， 导致无数的疾病用于纠正单倍不足的有效基因激活策略尚未被发现。 因为抑制蛋白质产生的机制尚不清楚。 反义寡核苷酸（ASO）是小的、可预测的和可编程的工具，其可以通过化学方法被修饰。 它们被设计成直接控制RNA的稳定性、加工和翻译，使它们可用于 剖析蛋白质生产的机制。以前在野生型细胞中的工作表明，空间阻断 反义寡核苷酸可以阻断核糖体上的替代翻译起始位点和直接剪接因子，以增加蛋白质的表达。 程度.此外，含有DNA的中心区域的阿索“缺口聚物”，其侧翼是化学修饰的寡核苷酸。 核苷酸，可以降解负调节蛋白质表达的RNA（例如反义转录物）。然而， 尚未研究这些策略在单倍不足情况下的功效。 在Jonathan Watts博士（阿索合成和化学）的指导下，合作者：Athma派博士 (RNA Anastasia Khvorova博士（阿索递送和神经生物学）和Dr. Xandra Breakefield（肿瘤抑制综合征），该提案旨在设计和应用化学- 修饰的ASO系统地研究内源性蛋白质阻遏机制，并确定关键的 调节全长蛋白质翻译的因素，使用NF 1基因作为模型。NF 1是一种肿瘤抑制因子 抑制Ras/MAPK信号传导。NF 1单倍不足导致1型神经纤维瘤病，一种遗传性疾病 其特征在于不受控制的神经细胞增殖和其它并发症。NF 1基因座是一个很好的 因为它有两个可供选择的翻译起始点-上游开放式阅读 在成熟mRNA的5'非翻译区（UTR）中有一个uORFs;与几个反义核酸重叠。 转录本;并且可能经历非生产性剪接。结合NF 1 5 'UTR uORF的空间阻断性ASO 已经合成，并确定了有希望的线索。目标1将测试这些阿索电极导线的有效性， 在主要起始位点翻译并增加蛋白质表达。Aim 2将设计和应用阿索间隙剂 靶向和降解NF 1反义转录物，并确定它们对NF 1蛋白表达的影响。目标3 将分离和测序NF 1新生RNA以鉴定隐蔽剪接位点。然后，ASO将被设计为阻止 这些位点并提高前体mRNA剪接效率。对于所有目标，候选ASO将被转染到 SH-SY 5 Y神经母细胞瘤细胞（其表达NF 1）用于批量筛选。成功的候选人将 在野生型和NF 1 +/-单倍不足神经元和雪旺细胞中进行了测试和优化。功能 通过测量Ras/MAPK活化来评估活化的NF 1蛋白。该项目将增加我们的 了解蛋白质表达是如何调节的，并可能为纠正单倍不足的策略提供信息。

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.