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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）和额颞叶痴呆（FTD）是密切相关的，致命的