Targeting Tau Splicing for Dementia

描述（由申请人提供）：淀粉样蛋白肽（A）和微管相关蛋白tau均与阿尔茨海默病（AD）的发病机制密切相关。然而，tau蛋白在AD发病机制中的作用尚未得到很好的理解，tau蛋白作为治疗靶点的作用还不充分。其致病作用的主要线索是tau基因中的近40个突变，这些突变导致某些形式的额颞叶痴呆（FTD），这是一种与AD相关的“tau蛋白病”。这些突变中的许多是沉默的，并改变tau前mRNA的剪接，导致外显子10的包含增加，从而改变含有3或4个微管结合结构域（分别为3R和4 R tau）的tau蛋白之间的平衡。我们已经验证了一个假设的茎环之间的边界外显子10和内含子10作为一个真正的结构，调节3R与4 R tau蛋白的形成细胞。我们还进行了高通量筛选，以确定结合并稳定这种茎环结构的小药物样化合物作为潜在的治疗策略，通过NMR确定与tau mRNA茎环结合的这些化合物之一的结构，并通过模拟设计验证这种结构模型。此外，我们已经开发了新的反义分子，识别不连续的mRNA序列，侧翼的tau前mRNA茎环结构和扭曲tau前mRNA剪接远离4 R亚型。这些反义分子是二分的，通过接头区连接，其中一部分与茎环上游的序列互补，另一部分与紧邻下游的序列互补。这种二分反义分子可以说是序列特异性和结构特异性的，因为同时结合两个侧翼区需要茎环形成。鉴于这些发现，我们现在提出将这两种方法联合收割机并开发反义MTX缀合物，其以高效力和选择性稳定tau茎环结构。因为MTX结合在茎的底部附近，接近反义分子结合的位点，所以将MTX连接到这样的反义分子预期产生高度有效和特异性的“分子扣”，其同时靶向tau前mRNA茎环结构、该结构上游的序列和该结构下游的序列。通过这些分子扣的这种三重结合的结果将是以治疗有益的方式有效和选择性地改变tau剪接。具体而言，我们建议：（1）产生反义-MTX缀合物，（2）测试这些缀合物结合并稳定tau前mRNA茎环结构的能力，和（3）确定这些缀合物使tau前mRNA的剪接远离4 R同种型的能力。

DESCRIPTION (provided by applicant): The amyloid-¿ peptide (A¿) and the microtubule-associated protein tau are both strongly implicated in the pathogenesis of Alzheimer's disease (AD). However, the role of tau in AD pathogenesis is not well understood, and tau is underdeveloped as a therapeutic target. Major clues to its pathogenic role are nearly 40 mutations in the tau gene that cause certain forms of frontotemporal dementia (FTD), a "tauopathy" related to AD. Many of these mutations are silent and alter the splicing of the tau pre-mRNA, leading to increased inclusion of exon 10 and thereby shifting the balance between tau proteins that contain 3 or 4 microtubule binding domains (3R and 4R tau, respectively). We have validated a postulated stem-loop at the boundary between exon 10 and intron 10 as a bona fide structure that regulates 3R versus 4R tau formation in cells. We have also carried out a high-throughput screen to identify small drug-like compounds that bind to and stabilize this stem-loop structure as a potential therapeutic strategy, determined the structure of one of these compounds bound to the tau mRNA stem-loop by NMR, and validated this structural model through analogue design. In addition, we have developed novel antisense molecules that recognize the discontinuous mRNA sequences that flank the tau pre-mRNA stem-loop structure and skew tau pre-mRNA splicing away from the 4R isoforms. These antisense molecules are bipartite, connected via a linker region, with one part complementary to the sequence just upstream from the stem-loop and the other complementary to the sequence immediately downstream. Such bipartite antisense molecules could be said to be both sequence- and structure-specific, as simultaneous binding to the two flanking regions requires stem-loop formation. In light of these findings, we now propose to combine these two approaches and develop antisense-MTX conjugates that stabilize the tau stem-loop structure with high potency and selectivity. Because MTX binds near the bottom of the stem, close to the sites where antisense molecules bind, linking MTX to such antisense molecules is expected to result in highly potent and specific "molecular clasps" that simultaneously target the tau pre-mRNA stem-loop structure, sequences upstream of this structure, and sequences downstream of this structure. The result of such tripartite binding by these molecular clasps would be potent and selective alteration of tau splicing in a therapeutically beneficial way. Specifically, we propose o: (1) generate antisense-MTX conjugates, (2) test these conjugates for their ability to bind to and stabilize the tau pre-mRNA stem-loop structure, and (3) determine the ability of these conjugates to shift splicing of the tau pre-mRNA away from the 4R isoforms.