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

肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD）是密切相关的，致命的 神经退行性疾病具有相当大的遗传和病理重叠。事后分析更多 超过97%的ALS病例和近一半的FTD病例显示细胞核清除和细胞质清除。 TDP-43的聚集，TDP-43是一种主要调节前体mRNA剪接的核DNA和RNA结合蛋白。 此外，大量证据表明，TDP-43核功能的丧失驱动了许多细胞的进化。 ALS/FTD发病机制方面。这项建议旨在建立在我们实验室最近的数据基础上，这些数据表明， RNA严格地将TDP-43束缚在细胞核内，并限制其被动核输出。我们建议设计一个 一种新的基于RNA的策略，以反对TDP-43核输出，由两个观察指导：1）TDP-43核 驻留取决于其与富含GU的核RNA的结合，和2）小的富含GU的RNA寡聚体转染到 活细胞有效地靶向和调节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（TNTAs），结合并保留游离的 TDP-43会扩散出细胞核。TNTA将由一个可逆的富含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 在稳定的核核糖核蛋白装配中的复合物。使用（GU）6序列作为起始点， 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 用于成功的TTS-TDP-43相互作用。细胞将通过合成的富含GU的RNA（或多聚A，作为RNA的载体）转染。 阴性对照），并且内源性TDP-43的核出口将通过免疫荧光监测， 自动共聚焦高内涵显微镜。TTS-TDP-43的直接相互作用，其对TDP-43的选择性， 43，然后评估随时间的可逆性以识别最佳TTS序列。第二个目标， 将测试几类内源性核RNA的核保留序列，以鉴定最佳的 NTS序列，当与TTS组合时可以促进TDP-43核定位。核束缚 通过噬菌体MS 2-环RNA-MCP系统的TNTAs将被用作阳性对照。TNTAs 然后将与富含GU的TTS连接的内源性经验证的NTS组合，测试其在预防 TDP-43出口。最后，将确定TNTAs对TDP-43的选择性和可逆性，并将确定TNTAs对TDP-43的选择性和可逆性。 将评价外显子掺入以确保不干扰TDP-43核功能。所有这些 研究将确定和优化一种新的工具，以促进TDP-43核定位，使进一步的治疗， 开发和提高我们对TDP-43核/胞质穿梭调节的理解。

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.