Restoring neurofilaments to axons in a mouse model of CMT2E

在 CMT2E 小鼠模型中将神经丝恢复为轴突

• 批准号: 10354366
• 负责人: Anthony Brown
• 金额: $ 43.31万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10354366
• 关键词: Address; Affect; Afferent Neurons; Age; Axon; Biological Assay; Caliber; Cellular biology; Charcot-Marie-Tooth Disease; Code; Collaborations; Data; Defect; Development; Disease; Electron Microscopy; Electrophysiology (science); Exhibits; Filament; Future; Genes; Goals; Impairment; Inherited; Injections; Interdisciplinary Study; Knock-in; Knock-out; Logic; Longitudinal Studies; Measurable; Missense Mutation; Modeling; Morphology; Motor Neurons; Mus; Mutation; Neural Conduction; Neurodegenerative Disorders; Neurons; Ohio; Outcome; Patients; Pediatric Hospitals; Peripheral; Peripheral Nerves; Physiological; Polymers; Proteins; Recombinant adeno-associated virus (rAAV); Research; Research Proposals; Structure; Testing; Therapeutic; Time; Translating; Universities; Viral Genes; Viral Vector; adeno-associated viral vector; design; dosage; feasibility testing; functional improvement; gene therapy; gene therapy clinical study; light microscopy; mouse genetics; mouse model; nerve conduction study; neurofilament; neurofilament protein L; neuromuscular function; null mutation; pre-clinical; restoration; success; vector

ABSTRACT This is a new R21 exploratory research proposal to establish proof-of-concept of a pre-clinical therapeutic strategy for Charcot-Marie-Tooth disease type 2E (CMT2E). CMT2E is caused by mutations in the NEFL gene, which codes for neurofilament protein L (NEFL). NEFL is a subunit of neurofilaments (NFs), which are cytoskeletal polymers that function to expand axon caliber and thereby increase axonal conduction velocity. An interesting feature of CMT2E is that it can be caused by null mutations (inherited recessively) or missense mutations (inherited dominantly). Preliminary data indicate that the cellular mechanisms of recessive and dominant CMT2E converge on an absence of NFs in peripheral myelinated axons, resulting in reduced axon caliber and nerve conduction defects. Our long-term goal is to develop a gene therapy strategy for restoring NFs to these NF-deficient axons. The central premise is that this will rescue axon caliber and nerve conduction and thereby ameliorate the disease. As a first step, we propose to test the feasibility of this strategy in the NEFL- /- mouse, which is a model of recessive CMT2E caused by a complete absence of the NEFL protein. Preliminary data demonstrate that NEFL-/- mice exhibit profound impairment of peripheral nerve conduction by 4 weeks of age or earlier. The logic in this mouse model is simple: these mice lack NFs and we seek to replace them. To achieve this goal, we propose a multi-disciplinary collaboration between four PIs at Ohio State University and Nationwide Children’s Hospital with deep and complementary expertise on the cell biology of NFs, viral gene therapy, neurodegenerative disease and electrophysiological assessments of neuromuscular function, including proven success in preclinical and clinical gene therapy studies. In Aim 1, we will test the hypothesis that expression of NEFL protein in neurons of NEFL-/- mice will restore NFs to the NF-deficient axons and thus increase axonal caliber. We will deliver NEFL to neurons of P0 NEFL-/- mice by intra-cerebroventricular injection of a recombinant adeno-associated virus. In Aim 2 we will test the hypothesis that the restoration of NFs to axons of NEFL-/- mice will rescue peripheral nerve conduction. The proposed research will address the following fundamental questions: (1) can we express NEFL protein efficiently in motor and sensory neurons, (2) does the NEFL protein assemble into NF polymers (3) are the NF polymers transported into the axons of peripheral nerves and over what time course, (4) does this result in partial or complete restoration of myelinated axon caliber and G-ratio, and (5) does this restore axonal nerve conduction? Successful completion of these aims will establish a proof-of-concept that NFs can be delivered to NF-deficient axons to rescue axon morphology and physiological function. This will lay the groundwork for future development of a general strategy both for dominant and recessive CMT2E, offering hope to patients with this intractable disease.

抽象的 这是一项新的R21探索性研究建议，旨在建立临床前疗法的概念验证 Charcot-Marie-Tooth疾病2e型（CMT2E）的策略。 CMT2E是由NEFL基因突变引起的， 编码神经丝蛋白L（NEFL）。 NEFL是神经丝（NFS）的亚基， 可膨胀轴突口径并因此增加轴突传导速度的细胞骨架聚合物。一个 CMT2E的有趣特征是，它可能是由无效突变引起的（遗传）或错义引起的 突变（主要继承）。初步数据，表明隐性和隐性的细胞机制 主要的CMT2E收敛于外周髓芯轴突中不存在NF，导致轴突减少 口径和神经传导缺陷。我们的长期目标是制定恢复基因疗法策略 NFS到这些NF缺陷轴突。中心前提是，这将挽救轴突口径和神经传导 从而改善疾病。作为第一步，我们建议测试该策略在NEFL中的可行性 / - 小鼠，这是由NEFL蛋白完全不存在引起的隐性CMT2E模型。初步的 数据表明，NEFL - / - 小鼠在4周内表现出对周围神经传导的严重损害 年龄或更早。该鼠标模型中的逻辑很简单：这些小鼠缺乏NFS，我们试图替换它们。到 实现这一目标，我们提出了俄亥俄州立大学四个PI之间的多学科合作 全国儿童医院具有NFS，病毒基因的细胞生物学的深刻而完整的专业知识 治疗，神经退行性疾病和神经肌肉功能的电生理评估，包括 在临床前和临床基因疗法研究中取得了验证的成功。在AIM 1中，我们将检验以下假设 NEFL蛋白在NEFL - / - 小鼠的神经元中的表达将恢复NF缺乏NF轴突，从而将NF恢复为 增加轴突口径。我们将通过脑室内注射将NEFL传递给P0 NEFL - / - 小鼠的神经元 重组腺相关病毒的。在AIM 2中，我们将检验以下假设： NEFL - / - 小鼠的轴突将挽救周围神经传导。拟议的研究将解决以下 基本问题：（1）我们可以在运动和感觉神经元中有效地表达NEFL蛋白吗？（2） NEFL蛋白组装成NF聚合物（3）是转运到外周神经轴突的NF聚合物 在什么时间课程中，（4）这会导致部分或完全恢复髓鞘的轴突口径和 G-Ratio，（5）这是否恢复轴突神经传导？这些目标的成功完成将建立 概念证明NF可以将NF递送到缺乏NF的轴突以营救轴突的形态和生理 功能。这将为未来发展的一般战略奠定基础 隐性CMT2E，为这种顽固性疾病的患者提供希望。