Targeting Tau Splicing for Dementia

描述（由申请人提供）：淀粉样蛋白肽（AQ）和微管相关蛋白tau都与阿尔茨海默病（AD）的发病机制密切相关。然而，tau 在 AD 发病机制中的作用尚不清楚，tau 作为治疗靶点也尚未开发出来。其致病作用的主要线索是 tau 基因中的近 40 个突变，这些突变会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，并改变 tau 前 mRNA 的剪接，导致外显子 10 的包含增加，从而改变包含 3 或 4 个微管结合域（分别为 3R 和 4R tau）的 tau 蛋白之间的平衡。我们已经验证了外显子 10 和内含子 10 之间边界处的假设茎环是调节细胞中 3R 与 4R tau 形成的真正结构。我们还进行了高通量筛选，以识别与 tau mRNA 茎环结构结合并稳定的小药物样化合物，作为潜在的治疗策略，通过 NMR 确定了其中一种与 tau mRNA 茎环结合的化合物的结构，并通过类似物设计验证了该结构模型。此外，我们还开发了新型反义分子，可以识别 tau 前体 mRNA 茎环结构侧翼的不连续 mRNA 序列，并使 tau 前体 mRNA 剪接偏离 4R 同工型。这些反义分子是二分的，通过接头区域连接，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，因为同时结合到两个侧翼区域需要形成茎环。鉴于这些发现，我们现在建议将这两种方法结合起来，开发反义 MTX 缀合物，以高效和选择性稳定 tau 茎环结构。由于 MTX 结合在茎底部附近，靠近反义分子结合的位点，因此将 MTX 连接到此类反义分子预计会产生高效且特异的“分子扣”，同时靶向 tau 前 mRNA 茎环结构、该结构的上游序列和该结构的下游序列。这些分子扣的三方结合的结果将以治疗有益的方式有效且选择性地改变 tau 剪接。具体来说，我们建议：(1) 生成反义-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，以及 (3) 确定这些缀合物将 tau 前 mRNA 剪接远离 4R 同工型的能力。

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.