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

项目摘要/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。在小鼠中，tau的聚集推动了疾病的进展，而 消耗tau会导致认知能力的改善，这表明靶向tau也可能使人类患者受益。一个 主要问题是如何实现这一目标。CRISPR技术(集群式规则间隔短 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 耗尽神经元中的tau。通过将不同的tau亚型与感兴趣的位点特定的引导RNA打靶，可以 可以想象的是，靶向存在于3R-tau病(例如，Pick病)、4R中的特定tau变体 肌萎缩侧索硬化症(如皮质基综合征)或混合型3R/4R肌萎缩侧索硬化症(如AD患者)。因此，我们转向了 一种新开发的CRISPR/Cas13技术，它允许靶向和耗尽RNA转录本 后者可能会对基因组产生有害的影响，包括DNA损伤，甚至 肿瘤发生学。CRISPR/Cas13技术可以为治疗性tau减少和 有可能克服困扰tau耗竭策略的许多限制。事实上，我们的初步调查 研究结果表明，我们可以通过计算预测哪些向导将成功地瞄准并耗尽tau，以及 我们在人类细胞系和原代人类神经元中展示了这一点，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来 开发一种用于神经系统(CNS)tau基因治疗的有效、非侵入性工具。在AIM-1中，我们将 开发和改进CRISPR/CAS13系统以针对tau，并将全面确定最优的 引导靶向3R-tau、4R-tau或总tau的RNA。我们将把这些指南传递给神经元和 确定其恢复正常神经功能的有效性和能力。在AIM-2中，我们将表演 进行临床前疗效测试，并确定我们最理想的CRISPR/Cas13复合体是否具有 阿尔茨海默病模型小鼠的治疗潜力。通过使用脑渗透剂在外围输送tau靶向导引 AAV方法，我们将通过进行一系列生化、组织学、 以及疾病进展的早期(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意义，因为它将开发针对tau所需的CRISPR技术，以及 此外，先进的基因治疗方法可以耗尽人类的tau，包括阿尔茨海默病和其他痴呆症患者。 其明显的含义是，瞄准并降低tau水平，即使是适度的，也可以提供一种新的治疗方法。 痴呆症患者的生活方式。

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.