Developing Remyelination Strategies for Demyelinating Optic Neuropathies Using Human Pluripotent Stem Cells

利用人类多能干细胞制定治疗脱髓鞘性视神经病的髓鞘再生策略

• 批准号: 10395288
• 负责人: Xitiz Chamling
• 金额: $ 8.89万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10395288
• 关键词: Acute; Advisory Committees; Apoptosis; Axon; Biology; Biometry; Blindness; CRISPR interference; Cells; Chronic; Coculture Techniques; Collaborations; Data; Demyelinating Diseases; Demyelinations; Disease; Environment; Genes; Goals; Human; In Vitro; Incidence; Inflammation; Institution; Laboratory Research; Learning; Mentors; MicroRNAs; Molecular; Multiple Sclerosis; Neural Conduction; Neurons; Oligodendroglia; Optic Nerve; Optic Neuritis; Pathway Analysis; Pathway interactions; Patients; Phase; Process; Reporter; Research; Retinal Ganglion Cells; Role; Scientist; Signal Pathway; System; Techniques; Training; Validation; Vision; Visual Acuity; Work; axon injury; base; career development; effective therapy; genome editing; high throughput screening; human pluripotent stem cell; human stem cells; innovation; multiple sclerosis patient; myelination; neuron loss; oligodendrocyte myelination; optic nerve disorder; remyelination; screening; single-cell RNA sequencing; small molecule; symposium; tool; transcription factor

PROJECT SUMMARY/ABSTRACT Optic nerve demyelinating diseases such as Optic Neuritis (ON) cause acute reduction of visual acuity and often chronic visual loss. The estimated incidence of ON in the USA is 6.4/100,000, and 50% of the 2.5 million multiple sclerosis (MS) patients worldwide are estimated to develop one or more episodes of ON during the course of their disease. Myelination of the optic nerve is carried out by a specialized cell, the oligodendrocyte (OL), that coats and protects axons and promotes neural conduction. Demyelination can cause apoptosis of the OLs, axonal damage, and neuronal cell death. Remyelination-based treatments have the potential to help protect retinal ganglion cell (RGC) neurons and reduce chronic vision loss, and perhaps help restore lost vision. The long-term goal of this project is to identify molecular pathways involved in differentiation and maturation of OLs that can be exploited for promoting remyelination of the demyelinated optic nerve. To this end, the PI has developed human pluripotent stem cell (hPSC) reporters for RGCs and OLs, and performed single cell RNA- sequencing and a preliminary high-throughput screen (HTS) to identify regulators of OL differentiation and maturation. Based upon this preliminary data, the PI proposes to: 1) expand the screening effort to identify additional compounds and pathways involved in OL maturation, 2) examine the transcription factors and pathways identified from the preliminary work for their role in OL differentiation, and 3) further identify and systemically characterize microRNAs (miRNAs) involved in OL specification and maturation. To perform the proposed studies, the PI will develop innovative tools such as hPSC-reporters for OL differentiation with inducible CRISPRi (interference) and CRISPRa (activation) system, and a functional in-vitro hOL/hRGC co-culture system for assessing hRGC myelination by hOLs. In the mentored phase, in Dr. Don Zack’s lab, the PI will carry out genome-editing to generate the CRISPRi and CRISPRa lines and acquire training at Wilmer’s HCS facility where small molecule screening to probe for signaling pathways involved in OL maturation will be performed. In the second year, under the guidance of Dr. Jay Baraban (an expert in microRNA biology), the miRNA studies on OLs will be initiated. The mentored phase will also be supplemented by training with Ingo Ruczinski (biostatistics collaborator), who will provide assistance with the pathway analysis and hit validation of HTS. Furthermore, the PI’s co-mentor, Dr. Peter Calabresi, who is a leading scientist in demyelinating diseases, will regularly meet, advise and help him prioritize the genes and pathways for further examination. Additionally, during the mentored phase, the PI will regularly meet with his advisory committee, attend scientific conferences, and continue his career development. The PI is in an ideal environment for the proposed research and for his career development as Dr. Zack has an established hPSC lab, state-of-the-art HCS facilities, and collaborations with renowned neuroscientists and vision-scientists. This will help PI to set-up good collaborations, learn new techniques, and build an independent research laboratory at a well-established academic institution.

项目摘要/摘要 视神经脱髓鞘疾病，肌肉神经炎（ON）引起视力急性的急性修复 慢性视觉损失。 据估计，在全球范围内的硬化症患者（MS）患者在此期间会发育一张或多个发作。 它们的疾病。 外套并保护轴突并促进神经脱髓鞘。 轴突损伤和神经元细胞死亡。 视网膜神经节细胞（RGC）神经元减少慢性视力丧失，也许有助于恢复视力 该项目的长期目标是确定与OLS的分化和成熟有关的分子途径 可以利用用于促进脱髓鞘神经的再生。 开发了用于RGC和OLS的人类多能干细胞（HPSC）记者，并进行了单细胞RNA- 测序和初步的高通量屏幕（HTS），以识别OL差异和 成熟。根据此初步数据，PI提出了：1） OL成熟涉及的其他化合物和途径，2）检查转录因子和 从初步工作中确定的途径。 系统地表征了涉及OL规范和成熟的microRNA（miRNA）。 支撑研究，PI将开发创新的工具，例如ASC替代者，以抗诱导 CRISPRI（干扰）和CRISPRA（激活）系统，以及功能性的Vitroro HOL/HRGC共文化系统 在指导阶段，在Don Zack博士的实验室中评估HRGC髓鞘 基因组编辑，以生成CRISPRI和CRISPRA系列，并在Wilmer的HCS设施中获得培训 将在您中进行小分子筛选到探测中涉及的信号通路 第二年，在Jay Baraban博士（MicroRNA生物学专家）的指导下，miRNA研究 OLS将被启动。 合作者），他将在途径分析并命中热的验证方面提供帮助。 PI的副赋予人Peter Calabresi博士是脱髓鞘疾病的主要科学家，将定期见面， 在指导的过程中，建议并帮助他优先考虑基因和途径。 阶段，PI将定期与他的咨询委员会会面，参加科学会议，并继续他的 职业发展。 作为Zack Hus博士建立的HPSC实验室，最先进的HCS设施以及与著名的合作 神经科学家和视觉科学家。 建立一个独立的研究实验室