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: 9979737
• 负责人: Gilbert Gallardo
• 金额: $ 39.35万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979737
• 关键词: Adult; Alzheimer' s Disease; Antibodies; Autophagocytosis; Blood - brain barrier anatomy; Brain; Bypass; Cells; 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; Neuraxis; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Onset of illness; Passive Immunization; Passive Immunotherapy; Pathogenesis; Pathologic; Pathology; Pharmacology; Plasma; Play; Proteins; RNA Interference; Role; Site; Tauopathies; Therapeutic; Transgenic Mice; Translating; Ubiquitin; aged; blood-brain barrier crossing; extracellular; improved; inhibitor/antagonist; innovation; intraperitoneal; intravenous administration; intravenous injection; knock-down; multicatalytic endopeptidase complex; neurotoxicity; prevent; protein degradation; self assembly; tau Proteins; tau expression; tau-1

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.

项目总结/摘要 病理性tau蛋白的积累是阿尔茨海默病中神经元缠结的主要成分， 几种退行性疾病，称为tau蛋白病。我们以前发现，给予抗- 针对人tau的tau抗体（h-tau）或抗tau分泌的单链可变片段（scFv）在 h-tau转基因小鼠（P301 S-tg）的中枢神经系统（CNS）降低，但没有去除所有的tau- 相关病理学虽然这些和其他研究证明了靶向tau蛋白的免疫方法 可以影响tau的发病机制，常规的免疫方法存在一些局限性， 在靶向tau蛋白病时排除了它们的全部潜力。包括，免疫治疗仅限于靶向 然而，大多数病理性tau保留在细胞的胞质溶胶中，而不是典型地 可接近细胞外抗体或分泌的scFv。此外，被动式的一个重要局限性 免疫是长期施用抗体的必要条件，其中只有很小的%（0.1-0.2%）交叉 血脑屏障（BBB）。当转化为人类时，这可能受到制造业的限制。以潜在地 克服这些局限性，我们假设抗tau单链抗体在神经元胞质溶胶中表达， （胞内抗体）将以改善的功效减少tau蛋白病。为了增强传统胞内抗体的独创性， 我们已经工程化了融合到泛蛋白的嵌合抗tau胞内抗体，其具有不同的突变或含有 热休克基序（HSC），其目的是穿梭细胞内tau以被蛋白酶体降解， 溶酶体或分子伴侣介导的自噬（CMA）。在初步数据中，表达修饰的抗tau蛋白 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 疾病发作后老年P301 S-tg小鼠中的修饰的抗tau胞内抗体有效地减少了tau蛋白病 而不含标签的常规抗tau胞内抗体在减少tau蛋白病方面无效。的目标 验证抗tau胞内抗体降低tau水平的降解机制 并确定抗tau胞内抗体预防或阻止tau蛋白病的程度。此外，我们的目标是绕过 通过将我们的抗tau胞内抗体与腺相关病毒介导的 通过静脉内给药在成年小鼠CNS中提供全局神经元转导的基因转移。 我们进一步提出产生靶向异常磷酸化tau位点的新的抗tau胞内抗体， 可以通过选择性降解病理性tau而显示出增强的功效。目前的提案利用了 胞内抗体的强度，其可用于靶向特定结构域或具有细胞内在免疫原性的修饰。 调节蛋白质降解的机制可能提供一种新的免疫抑制剂， 提高功效。