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: 10527601
• 负责人: Xitiz Chamling
• 金额: $ 24.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527601
• 关键词: Acute; Advisory Committees; Apoptosis; Astrocytes; Axon; Biological Assay; Biology; Biometry; Blindness; CRISPR interference; CRISPR-mediated transcriptional activation; Candidate Disease Gene; Cell Line; Cells; Chronic; Coculture Techniques; Collaborations; Complement; DICER1 gene; Data; Demyelinating Diseases; Demyelinations; Disease; Environment; Enzymes; Epidermal Growth Factor Receptor; Genes; Goals; Human; Image; In Vitro; Incidence; Inflammation; Inflammatory; Institution; Laboratory Research; Lead; Learning; MAP2K1 gene; MAPK3 gene; MEKs; Mentors; MicroRNAs; Molecular; Multiple Sclerosis; Mus; Myelin Basic Proteins; Neural Conduction; Neurons; Oligodendroglia; Optic Nerve; Optic Neuritis; Pathway Analysis; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phosphotransferases; Play; Population; Process; Proteins; Proteolipids; Publishing; Reporter; Reporting; Research; Research Personnel; Retinal Ganglion Cells; Role; SOX5 gene; SOX6 gene; Scientist; Signal Pathway; System; Techniques; Training; Validation; Vision; Visual Acuity; Work; axon injury; base; career development; data tools; differentiation protocol; effective therapy; gain of function; genome editing; high throughput screening; human pluripotent stem cell; human stem cells; innovation; insight; miRNA expression profiling; multiple sclerosis patient; myelination; nanoluciferase; neuron loss; neuroprotection; novel; oligodendrocyte lineage; oligodendrocyte myelination; oligodendrocyte precursor; optic nerve disorder; precursor cell; programs; remyelination; screening; single-cell RNA sequencing; small molecule; stem cell differentiation; stem cells; 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，如视神经炎(ON)，会导致视力急剧下降，通常 慢性视力丧失。据估计，美国ON的发病率为6.4/10万，是250万人中的50% 据估计，全世界的硬化症(MS)患者在以下过程中会发生一次或多次ON 他们的病。视神经的髓鞘形成是由一种特殊的细胞--少突胶质细胞(OL)执行的，这种细胞 包裹和保护轴突，促进神经传导。脱髓鞘可导致白斑细胞的凋亡， 轴突损伤和神经细胞死亡。以髓鞘再生为基础的治疗有可能帮助保护 视网膜神经节细胞(RGC)神经元和减少慢性视力丧失，也许有助于恢复视力。这个 该项目的长期目标是确定与OL分化和成熟有关的分子途径。 这可以被用来促进脱髓鞘视神经的重新髓鞘形成。为此，国际刑警组织有 建立了人多能干细胞(HPSC)报告RGC和OLS，并进行了单细胞RNA- 测序和初步高通量筛选(HTS)以确定OL分化和 成熟。根据这一初步数据，PI建议：1)扩大筛查工作，以确定 参与OL成熟的其他化合物和途径，2)检测转录因子和 从初步工作中确定的在OL分化中的作用的途径，以及3)进一步确定和 系统地描述与OL规范和成熟有关的microRNAs(MiRNAs)。要执行以下操作 拟议的研究，PI将开发创新工具，如hPSC-Reports，用于与Inducable进行OL差异化 CRISPRI(干扰)和CRISPRA(激活)系统，以及体外Hol/hRGC共培养系统 用于评估hRGC的HALS髓鞘形成。在指导阶段，在唐·扎克博士的实验室里，PI将执行 基因组编辑以产生CRISPRi和CRISPRa系并在Wilmer的HCS设施获得培训 将进行小分子筛选，以探索与OL成熟有关的信号通路。在 第二年，在Jay Baraban博士(microRNA生物学专家)的指导下，miRNA研究 将启动OL。指导阶段还将辅以Ingo Ruczinski(生物统计学)培训 协作者)，他将为HTS的路径分析和HIT验证提供帮助。此外， 少年派的共同导师彼得·卡拉布雷西博士是脱髓鞘疾病的领先科学家，他将定期会面， 建议并帮助他确定进一步检查的基因和途径的优先顺序。此外，在接受指导的 在这一阶段，私人投资委员会将定期与他的顾问委员会会面，出席科学会议，并继续他的 职业发展。对于提议的研究和他的职业发展，PI处于一个理想的环境中 因为扎克博士拥有成熟的hPSC实验室、最先进的HCS设施，并与著名的 神经学家和视觉科学家。这将帮助PI建立良好的合作关系，学习新技术，并 在成熟的学术机构建立独立的研究实验室。