Engineering Anti-Tau Intrabodies that Reduce Tauopathy by Either the Proteasome, Lysosome, or Chaperone Mediated Autophagy

项目摘要/摘要 病理性tau的积聚是阿尔茨海默病和老年痴呆症神经原纤维缠结的主要成分。 几种退行性疾病，被称为tauopathy。我们之前发现，政府的反- 人tau抗体(h-tau)或分泌抗tau单链可变区(ScFv)的表达 H-tau转基因小鼠(P301S-TG)的中枢神经系统(CNS)减少，但不能全部清除tau-Tg。 相关病理学。虽然这些和其他研究表明针对tau的免疫治疗方法 可以影响tau的发病机制，传统的免疫治疗方法存在一些局限性 排除了他们的全部潜力，当目标是直肠痉挛。包括，免疫治疗仅限于靶向治疗 细胞外蛋白，而大多数病理tau保留在细胞胞浆中，通常不是 可被胞外抗体或分泌的单链抗体所访问。此外，被动的一个重要限制是 免疫是慢性注射抗体所必需的，其中只有一小部分(0.1-0.2%)交叉 血脑屏障(BBB)。当转化为人类时，这可能会受到制造业的限制。潜在地 克服这些限制，我们假设抗tau单链抗体在神经元胞浆中的表达。 (体内)将减少紧张症，并提高疗效。为了增强传统体内的独创性， 我们已经设计了嵌合的反tau抗体，融合到泛素上，含有不同的突变或含有 热休克基序(HSC)，其目标是穿梭于细胞内的tau被蛋白酶体、 溶酶体或伴侣介导的自噬(CMA)。在初步数据中，表达修饰的反式tau 在原代培养的神经元中，表达h-tau的体内h-tau蛋白水平降低。此外，该表达式 经修饰的抗tau抗体在老年P301S-TG小鼠发病后有效地减轻了tau病 然而，常规的不含标签的抗tau抗体在减少tau病方面是无效的。目标 这个项目的目的是验证抗tau抗体体内降低tau水平的降解机制。 并确定体内抗tau抗体可在多大程度上预防或阻止tau病。此外，我们的目标是绕过 我们的抗tau抗体与腺相关病毒介导的最新进展相结合的血脑屏障 通过静脉给药在成年小鼠中枢神经系统中提供全局神经元转导的基因转移。 我们进一步建议产生新的针对异常磷酸化tau位点的抗tau抗体， 可能通过选择性地降解病理性tau而显示出增强的疗效。目前的提案利用了 体内的强度，可针对特定的结构域或使用细胞固有的修饰进行修改 调节蛋白质降解的机制可能提供一种新的免疫治疗剂 提高了疗效。

Project Summary/Abstract The accumulation of pathological tau is the main component of neurofibrillary tangles in Alzheimer’s disease and several degenerative diseases, referred to as tauopathies. We previously found that administration of an anti- tau antibody to human tau (h-tau) or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system (CNS) of h-tau transgenic mice (P301S-tg) decreased but did not remove all tau- associated pathology. While these and other studies demonstrate immunotherapeutic approaches targeting tau can influence tau pathogenesis, conventional immunotherapeutic approaches present some limitations that preclude their full potential when targeting tauopathy. Including, immunotherapy is limited to targeting extracellular proteins whereas the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody or secreted scFv. In addition, a significant limitation of passive immunization is the necessity for chronic administration of antibodies of which only a small % (0.1-0.2%) cross the blood-brain-barrier (BBB). When translated into humans, this may be limited by manufacturing. To potentially overcoming these limitations, we hypothesize the expression of anti-tau scFv in the cytosol of neurons (intrabodies) will reduce tauopathy with improved efficacy. To enhance the ingenuity of conventional intrabodies, we have engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations or containing a heat-shock motif (HSC) with the goal of shuttling intracellular tau for degradation by either the proteasome, lysosome or chaperone-mediated autophagy (CMA). In preliminary data, expressing the modified anti-tau intrabodies in primary neuronal cultures expressing h-tau reduced h-tau protein levels. Moreover, the expression of the modified anti-tau intrabodies in aged P301S-tg mice after disease onset effectively reduced tauopathy whereas; a conventional anti-tau intrabody containing no tags was ineffective in reducing tauopathy. The goals for this project are to validate the degradation mechanisms by which the anti-tau intrabodies reduce tau levels and determine the extent to which anti-tau intrabodies prevent or stop tauopathy. In addition, we aim at bypass the BBB by combining our anti-tau intrabodies with the recent advances in adeno-associated virus-mediated gene transfer that provide global-neuronal transduction in the adult mouse CNS by intravenous administration. We further propose to generate new anti-tau intrabodies that target aberrant phosphorylated tau sites, which may display an enhanced efficacy by selectively degrading pathological tau. The current proposal harnessed the strength of intrabodies, which are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation potentially providing a new immunotherapeutic agent with improved efficacy.