Development of TDP-43 nuclear targeting aptamers for ALS/FTD

用于 ALS/FTD 的 TDP-43 核靶向适体的开发

• 批准号: 10558610
• 负责人: Lindsey Renae Hayes
• 金额: $ 8.19万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10558610
• 关键词: Amyotrophic Lateral Sclerosis; Attenuated; Autopsy; Benchmarking; Binding; Biological Assay; Broccoli - dietary; Cell Nucleus; Cells; Chemistry; Chromatin; Complex; Cytoplasm; DNA Binding; Data; Development; Diffuse; Disease; Dissociation; Dose; Doxycycline; Ensure; Enterobacteria phage MS2; Evaluation; Evolution; Exons; Frontotemporal Dementia; Future; Genetic; Genetic Transcription; Hela Cells; Histone H2B; Histone H3; Histone H4; Immunofluorescence Immunologic; Immunoprecipitation; Intervention; Laboratories; Length; Link; MALAT1 gene; MS2 coat protein; Mass Spectrum Analysis; Mediating; Messenger RNA; MicroRNAs; Microscopy; Monitor; Mutation; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Export; Nuclear RNA; Nucleotides; Pathogenesis; Pathogenicity; Pathologic; Patients; Plasmids; Poly A; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Regulation; Research; Ribonucleoproteins; Small Nucleolar RNA; Structure; System; TDP-43 aggregation; Testing; Therapeutic; Time; Transfection; U6 small nuclear RNA; aptamer; arm; beta Globin; confocal imaging; crosslink; crosslinking and immunoprecipitation sequencing; design; effective therapy; frontotemporal lobar dementia amyotrophic lateral sclerosis; improved; inhibitor; insight; mRNA Precursor; novel; prevent; protein TDP-43; prototype; residence; therapy development; tool; vector

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are closely-related, fatal neurodegenerative diseases with considerable genetic and pathologic overlap. Postmortem analysis of more than 97% of ALS cases and nearly half of FTD cases demonstrates nuclear clearance and cytoplasmic aggregation of TDP-43, a primarily nuclear DNA- and RNA-binding protein that regulates pre-mRNA splicing. Moreover, abundant evidence shows that the loss of TDP-43 nuclear function drives the evolution of many aspects of ALS/FTD pathogenesis. This proposal seeks to build on recent data from our laboratory showing that RNA critically tethers TDP-43 within the nucleus and limits its passive nuclear export. We propose to design a novel RNA-based strategy to oppose TDP-43 nuclear export, guided by two observations: 1) TDP-43 nuclear residence depends on its binding to GU-rich nuclear RNAs, and 2) small GU-rich RNA oligomers transfected into live cells efficiently target and modulate the nuclear/cytoplasmic shuttling of TDP-43. Here, we will develop bivalent RNA oligomers, termed TDP-43 nuclear targeting aptamers (TNTAs), to bind and retain the pool of free TDP-43 that would otherwise diffuse out of the nucleus. The TNTA will consist of a reversible GU-rich TDP-43 targeting sequence (TTS), a linker, and a nucleus-targeting sequence (NTS), to anchor the TNTA-TDP-43 complex within stable nuclear ribonucleoprotein assemblies. Using the (GU)6 sequence as a starting point, studies in aim 1 will further optimize the TTS motif, using the TDP-43 nuclear displacement assay as a readout for successful TTS-TDP-43 interaction. Cells will be transfected by synthetic GU-rich RNAs (or poly-A, as a negative control), and the nuclear exit of endogenous TDP-43 will be monitored by immunofluorescence with automated confocal high-content microscopy. The direct TTS-TDP-43 interaction, its selectivity towards TDP- 43, and reversibility over time will then be evaluated to identify the optimal TTS sequence. In the second aim, the nuclear retention sequences of several classes of endogenous nuclear RNAs will be tested to identify optimal NTS sequences, that, when combined with the TTS can promote TDP-43 nuclear localization. Nuclear tethering of TNTAs via the bacteriophage MS2-loop RNA-MCP system will be utilized as a positive control. TNTAs combining the endogenous validated NTS linked to the GU-rich TTS will then be tested for activity in preventing TDP-43 exit. Finally, the selectivity and reversibility of the TNTAs towards TDP-43 will be determined, and cryptic exon incorporation will be evaluated to ensure no interference with TDP-43 nuclear function. Together, these studies will identify and optimize a new tool to promote TDP-43 nuclear localization, to enable further therapy development and improve our understanding of the regulation of TDP-43 nuclear/cytoplasmic shuttling.

肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 密切相关，致命 神经退行性疾病具有相当大的遗传和病理重叠。更多事后分析 超过 97% 的 ALS 病例和近一半的 FTD 病例表现出核清除和细胞质 TDP-43 的聚集，TDP-43 是一种主要的核 DNA 和 RNA 结合蛋白，调节前 mRNA 剪接。 此外，大量证据表明，TDP-43核功能的丧失驱动了许多物种的进化。 ALS/FTD 发病机制的各个方面。该提案旨在以我们实验室的最新数据为基础，表明 RNA 将 TDP-43 关键地束缚在细胞核内并限制其被动核输出。我们建议设计一个 基于 RNA 的新型策略来阻止 TDP-43 核输出，以两个观察为指导：1) TDP-43 核 停留取决于其与富含 GU 的核 RNA 的结合，以及 2) 转染到的富含 GU 的小 RNA 寡聚体 活细胞有效地靶向并调节 TDP-43 的核/细胞质穿梭。在这里，我们将开发 二价 RNA 寡聚体，称为 TDP-43 核靶向适体 (TNTA)，用于结合并保留游离核酸池 TDP-43否则会扩散出细胞核。 TNTA 将由富含 GU 的可逆 TDP-43 组成 靶向序列 (TTS)、接头和核靶向序列 (NTS)，用于锚定 TNTA-TDP-43 稳定核糖核蛋白组件内的复合物。使用 (GU)6 序列作为起点， 目标 1 的研究将进一步优化 TTS 基序，使用 TDP-43 核位移测定作为读数 成功的 TTS-TDP-43 交互。细胞将被合成的富含 GU 的 RNA（或聚 A，作为 阴性对照），内源性 TDP-43 的核退出将通过免疫荧光监测 自动化共焦高内涵显微镜。直接 TTS-TDP-43 相互作用，其对 TDP- 的选择性 43，然后将评估随时间的可逆性，以确定最佳 TTS 序列。在第二个目标中， 将测试几类内源性核 RNA 的核保留序列，以确定最佳的 NTS序列表明，当与TTS结合时可以促进TDP-43核定位。核束缚 通过噬菌体 MS2 环 RNA-MCP 系统的 TNTA 将用作阳性对照。 TNTA 将内源性验证的 NTS 与富含 GU 的 TTS 相结合，然后将测试其预防活性 TDP-43退出。最后，TNTA 对 TDP-43 的选择性和可逆性将被确定，并且神秘 将评估外显子掺入，以确保不干扰 TDP-43 核功能。在一起，这些 研究将确定并优化一种新工具来促进 TDP-43 核定位，以实现进一步的治疗 的发展并提高我们对 TDP-43 核/细胞质穿梭调节的理解。