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

肌萎缩侧索硬化症(ALS)和额颞痴呆(FTD)密切相关，是致命的 神经退行性疾病，有相当大的遗传和病理重叠。更多的尸检分析 超过97%的ALS病例和近一半的FTD病例显示胞核清除和胞浆 TDP-43的聚集，主要是一种核DNA和RNA结合蛋白，调节前mRNA剪接。 此外，大量证据表明，TDP-43核功能的丧失推动了许多 ALS/FTD发病机制的几个方面。这项建议旨在以我们实验室的最新数据为基础，表明 RNA关键地将TDP-43束缚在核内，并限制其被动核出口。我们建议设计一种 基于RNA的反对TDP-43核出口的新策略，由两个观察指导：1)TDP-43核 滞留取决于它与富含Gu的核RNA的结合，以及2)小的富含Gu的RNA寡聚体 活细胞有效地靶向并调节TDP-43的核/胞质穿梭。在这里，我们将发展 二价RNA低聚物，称为TDP-43核靶向适配子(TNTA)，用于结合和保留游离池 TDP-43，否则会扩散出细胞核。TNTA将由可逆的富含Gu的TDP-43组成 靶向序列(TTS)、接头和核靶向序列(NTS)，以锚定TNTA-TDP-43 稳定的核核糖核蛋白组件中的复合体。使用(GU)6序列作为起点， 目标1中的研究将进一步优化TTS基序，使用TDP-43核置换分析作为读数 以获得成功的TTS-TDP-43相互作用。细胞将被合成的富含GU的RNA(或PolyA，作为一种 阴性对照)，并用免疫荧光法监测内源性TDP-43的核退出 自动共聚焦高含量显微镜。TTS-TDP-43的直接相互作用及其对TDP-43的选择性 43，然后将评估随时间的可逆性以确定最佳的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 NTS序列，当与TTS结合时，可以促进TDP-43核定位。核系留 通过噬菌体MS2-loop RNA-MCP系统的TNTAs将被用作阳性对照。TNTAs 将内源性验证的NT与富含GU的TTS相结合，然后将测试其在预防 TDP-43出口。最后，TNTAs对TDP-43的选择性和可逆性将被确定，而且是隐晦的 将对外显子掺入进行评估，以确保不干扰TDP-43的核功能。加在一起，这些 研究将确定和优化一种新的工具来促进TDP-43核定位，以实现进一步的治疗 发展和提高我们对TDP-43核质穿梭调控的理解。

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.