Targeting Tau Splicing for Dementia

描述(申请人提供)：淀粉样肽(A？)和微管相关蛋白tau都与阿尔茨海默病(AD)的发病密切相关。然而，tau在AD发病机制中的作用还不是很清楚，并且tau作为一个治疗靶点还不发达。它的致病作用的主要线索是tau基因的近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病”。其中许多突变是沉默的，改变了tau前mRNA的剪接，导致外显子10的包含增加，从而改变了包含3或4个微管结合结构域(分别为3R和4R tau)的tau蛋白之间的平衡。我们已经验证了在外显子10和内含子10之间的边界上的假设的茎环是调节细胞中3R和4R tau形成的真正结构。我们还进行了高通量筛选，以确定与这种茎环结构结合并稳定的小分子类药物化合物作为一种潜在的治疗策略，通过核磁共振确定了其中一种与tau mRNA茎环结合的化合物的结构，并通过模拟设计验证了该结构模型。此外，我们还开发了新型的反义分子，可以识别tau前mRNA茎环结构侧翼的不连续mRNA序列，并使tau前mRNA与4R亚型的剪接偏离。这些反义分子是两部分的，通过连接区连接，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。这种两部分反义分子可以说既是序列特异性的，也是结构特异性的，因为同时与两个侧翼区域结合需要形成茎环。鉴于这些发现，我们现在建议将这两种方法结合起来，开发出反义-MTX结合物，以高效和选择性地稳定tau茎环结构。由于MTX结合在茎的底部附近，靠近反义分子结合的部位，将MTX与这些反义分子连接起来有望产生高度有效和特异的“分子夹”，同时靶向tau前mRNA茎环结构、该结构上游的序列和该结构的下游序列。这些分子卡环的这种三方结合的结果将是以一种有益于治疗的方式有效和选择性地改变tau剪接。具体地说，我们建议：(1)生成反义MTX结合物，(2)测试这些结合物与tau前mRNA茎环结构结合和稳定的能力，以及(3)确定这些结合物将tau前mRNA的剪接从4R亚型转移出去的能力。

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.