CRISPR gene therapies targeting tau in Alzheimer's disease and tauopathies

项目总结/摘要 编码tau蛋白的MAPT基因是阿尔茨海默病（AD）治疗的理想靶点，因为它 在所有AD患者中形成标志性病理学。在小鼠中，tau聚集驱动疾病进展，而 消耗tau蛋白导致认知改善，这表明靶向tau蛋白也可能有益于人类患者。一 主要问题是如何实现这一目标。CRISPR技术（CRISPR Regularly Interspaced Short） 回文重复）近年来蓬勃发展，我们现在可以利用这种方法来具体地 消耗神经元中的tau蛋白。通过用感兴趣的位点特异性引导RNA靶向不同的tau同种型，可以 可以想象靶向3R-tau蛋白病中存在的特定tau变体（例如，Pick病），4 R tau蛋白病（例如，皮质基底综合征），或混合型3R/4 R tau蛋白病（例如，AD患者）。于是，我们转向了 一种新开发的CRISPR/Cas 13技术，该技术允许靶向和耗尽RNA转录物， 而不是DNA本身，后者可能对基因组产生不必要的影响，包括DNA损伤， 肿瘤发生CRISPR/Cas 13技术可以为治疗性tau蛋白减少和 潜在地克服了困扰tau耗竭策略的许多限制。事实上，我们初步的 研究结果表明，我们可以通过计算预测哪些向导将成功靶向和耗尽tau蛋白， 我们在人类细胞系和原代人类神经元中证明了这一点，这表明我们已经找到了一种最佳的 用于指导预测、开发和验证的管道。因此，我们有一个令人兴奋的机会， 为神经系统（CNS）中的tau基因治疗开发有效的非侵入性工具。在目标1中，我们将 开发和完善CRISPR/Cas 13系统以靶向tau蛋白，并将全面确定最佳的 靶向3R-tau、4 R-tau或总tau的向导RNA。我们将把这些指南传递给神经元， 确定其有效性和恢复正常神经元功能的能力。在Aim-2中，我们将执行 临床前功效测试并确定我们最佳的CRISPR/Cas 13复合物是否具有 AD模型小鼠的治疗潜力。通过使用脑渗透剂在外周递送tau靶向引导物 AAV方法，我们将通过进行一系列生化，组织学， 以及在疾病进展的早期（预防）或晚期（治疗）阶段的行为研究。这 这项研究具有创新性和重要性，因为它将开发靶向tau蛋白所需的CRISPR技术， 还推进基因治疗方法，以消耗人类（包括AD和其他痴呆患者）中的tau蛋白。 明确的含义是，靶向和降低tau蛋白水平，即使是适度的，也可以提供一种新的治疗方法 痴呆症患者的治疗方法。

PROJECT SUMMARY / ABSTRACT The MAPT gene encoding the tau protein is an ideal target for Alzheimer’s disease (AD) therapeutics since it forms hallmark pathology in all AD patients. In mice, tau aggregation drives disease progression, while depleting tau leads to cognitive improvements, suggesting targeting tau may benefit human patients as well. A major question has been how to achieve this goal. CRISPR technology (Clustered Regularly Interspaced Short Palindromic Repeats) has blossomed in recent years, and we can now leverage this approach to specifically deplete tau in neurons. By targeting different tau isoforms with site-specific guide RNAs of interest, one can conceivably target the specific tau variants that are present in 3R-tauopathies (e.g., Pick’s disease), 4R tauopathies (e.g., corticobasal syndrome), or mixed 3R/4R tauopathies (e.g., AD patients). Thus, we turned to a newly developed CRISPR/Cas13 technology, which allows targeting and depletion of RNA transcripts rather than DNA itself, the latter of which can have unwanted effects on the genome including DNA damage and even tumorigenesis. CRISPR/Cas13 technology could provide new opportunities for therapeutic tau reduction and potentially overcome many of the limitations that have plagued tau depletion strategies. In fact, our preliminary findings show that we can computationally predict which guides will successfully target and deplete tau, and we demonstrated this in a human cell line and primary human neurons, suggesting we have found an optimal pipeline for guide prediction, development, and validation. Therefore, we have an exciting opportunity to develop an effective, non-invasive tool for tau gene therapy in the nervous system (CNS). In Aim-1, we will develop and refine the CRISPR/Cas13 system to target tau and will comprehensively identify the most optimal guide RNAs that target either 3R-tau, 4R-tau, or total tau. We will deliver those guides to neurons and determine their effectiveness and ability to restore normal neuronal function. In Aim-2, we will perform preclinical efficacy testing and determine whether our most optimal CRISPR/Cas13 complexes have therapeutic potential in AD model mice. By delivering tau-targeting guides peripherally using brain-penetrant AAV methods, we will evaluate their therapeutic impact by performing a battery of biochemical, histological, and behavioral studies at either early (preventative) or late (therapeutic) stages of disease progression. This study is innovative and significant since it will both develop the CRISPR technology needed to target tau, and also advance gene therapy approaches to deplete tau in humans including AD and other dementia patients. The clear implications are that targeting and reducing tau levels, even modestly, could provide a new treatment modality for dementia patients.