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

基本信息

批准号：
10427644
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
项目类别：
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/10427644
关键词：
Amyotrophic Lateral Sclerosis Attenuated Autopsy Benchmarking Binding Biological Assay Broccoli - dietary Cell Nucleus Cells Chemistry Chromatin Complex 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 Testing Therapeutic Time U6 small nuclear RNA aptamer arm base 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

项目摘要

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.

肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）密切相关，致命具有相当大的遗传和病理重叠的神经退行性疾病。验尸分析更多超过97％的ALS病例和近一半的FTD病例表明核清除率和细胞质 TDP-43的聚集是一种主要是调节前MRNA剪接的核DNA和RNA结合蛋白。此外，大量证据表明，TDP-43核功能的丧失推动了许多 ALS/FTD发病机理的各个方面。该建议旨在基于我们实验室的最新数据，表明 RNA对核内的TETHER TDP-43进行了关键，并限制了其被动核输出。我们建议设计一个基于RNA的新型策略反对TDP-43核出口，以两个观察为指导：1）TDP-43核居住取决于其与富含GU的核RNA的结合，2）转染的小GU RNA低聚物活细胞有效地靶向并调节TDP-43的核/细胞质穿梭。在这里，我们将发展二价RNA低聚物，称为TDP-43核靶向适体（TNTA），以结合和保留游离的库 TDP-43否则会从细胞核中扩散出来。 TNTA将由可逆的GU-RICH TDP-43组成靶向序列（TTS），接头和核定靶向序列（NTS），以锚定TNTA-TDP-43 稳定的核核糖核蛋白组件中的复合物。使用（gu）6序列作为起点， AIM 1中的研究将使用TDP-43核位移作为读数，进一步优化TTS基序成功进行TTS-TDP-43相互作用。细胞将通过富含GU的RNA（或Poly-A作为A）转染细胞阴性对照），内源性TDP-43的核出口将通过免疫荧光监测自动共聚焦高核显微镜。直接TTS-TDP-43相互作用，其对TDP-的选择性然后，将评估43，并且随着时间的推移可逆性，以识别最佳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，以鉴定最佳 NTS序列，与TT结合使用可以促进TDP-43核定位。核束缚通过噬菌体MS2-loop RNA-MCP系统的TNTA被用作阳性对照。 tntas 结合与富含GU的TT相关的内源性验证的NTS将进行测试以防止活性 TDP-43出口。最后，将确定TNTA对TDP-43的选择性和可逆性，并隐秘将评估外显子掺入，以确保对TDP-43核功能没有干扰。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开发并改善了我们对TDP-43核/细胞质穿梭的调节的理解。