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

工程抗 Tau 胞内抗体可通过蛋白酶体、溶酶体或伴侣介导的自噬减少 Tau 病

• 批准号: 10612351
• 负责人: Gilbert Gallardo
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612351
• 关键词: Adult; Alzheimer' s Disease; Antibodies; Autophagocytosis; Blood - brain barrier anatomy; Brain; Bypass; Cells; Central Nervous System; Chronic; Cytosol; Data; Degenerative Disorder; Dementia; Dependovirus; Disease; Engineering; Extracellular Protein; Gene Transfer; Goals; Heat-Shock Response; Human; Immunotherapeutic agent; Immunotherapy; Intrabody; Intravenous; Location; Lysosomes; Mediating; Microtubules; Modification; Molecular Chaperones; Mus; Mutation; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Onset of illness; Passive Immunization; Passive Immunotherapy; Pathogenesis; Pathologic; Pathology; Plasma; Proteins; RNA Interference; Site; Tauopathies; Therapeutic; Transgenic Mice; Translating; Ubiquitin; abnormally phosphorylated tau; aged; blood-brain barrier crossing; extracellular; improved; inhibitor; innovation; intraperitoneal; intravenous administration; intravenous injection; knock-down; manufacture; multicatalytic endopeptidase complex; neuroprotection; neurotoxicity; pharmacologic; prevent; protein degradation; self assembly; tau Proteins; tau expression

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.

项目概要/摘要 病理性 tau 蛋白的积累是阿尔茨海默病和阿尔茨海默病中神经原纤维缠结的主要成分 几种退行性疾病，称为 tau蛋白病。我们之前发现，施用抗 人 tau 抗体 (h-tau) 或抗 tau 分泌单链可变片段 (scFv) 的表达 h-tau 转基因小鼠 (P301S-tg) 的中枢神经系统 (CNS) 减少，但并未消除所有 tau- 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 可能影响 tau 发病机制，但传统的免疫治疗方法存在一些局限性 当针对 tau 蛋白病时，会阻碍其充分发挥潜力。其中，免疫疗法仅限于靶向 细胞外蛋白，而大多数病理性 tau 保留在细胞的胞浆中，通常不存在 可接触细胞外抗体或分泌的 scFv。此外，被动式的一个重大限制 免疫接种是长期施用抗体的必要条件，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，这可能会受到制造的限制。潜在地 克服这些限制，我们假设抗 tau scFv 在神经元细胞质中表达 （体内）将减少 tau 蛋白病变并提高疗效。为了增强传统体内的独创性， 我们设计了与泛素融合的嵌合抗 tau 抗体，其中含有不同的突变或含有 热休克基序 (HSC)，其目标是穿梭细胞内 tau 蛋白，以便被蛋白酶体降解， 溶酶体或分子伴侣介导的自噬（CMA）。在初步数据中，表达了修饰的 anti-tau 表达 h-tau 的原代神经元培养物中的胞内抗体降低了 h-tau 蛋白水平。此外，表达式 老年 P301S-tg 小鼠发病后修饰的抗 tau 胞内抗体有效减少了 tau 病 然而;不含标签的传统抗 tau 抗体胞内抗体对于减少 tau 蛋白病变无效。目标 该项目的目的是验证抗 tau 体内抗体降低 tau 水平的降解机制 并确定抗 tau 蛋白胞内抗体预防或阻止 tau 蛋白病变的程度。另外，我们的目标是绕过 BBB 通过将我们的抗 tau 抗体与腺相关病毒介导的最新进展相结合 通过静脉注射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。 我们进一步建议生成针对异常磷酸化 tau 位点的新抗 tau 胞内抗体， 可能通过选择性降解病理性 tau 蛋白而表现出增强的功效。当前的提案利用了 体内抗体的强度，可针对特定域或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 提高疗效。