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.

项目概要/摘要 视神经脱髓鞘疾病，例如视神经炎 (ON) 会导致视力急剧下降，并且常常会导致视敏度急剧下降。 在美国，慢性视力丧失的估计发病率为 6.4/100,000，占 250 万倍数的 50%。 据估计，全世界的硬化症 (MS) 患者在治疗过程中都会出现一次或多次 ON 发作。 他们的疾病是由一种特殊的细胞少突胶质细胞（OL）进行的。 覆盖并保护轴突并促进神经传导，脱髓鞘可导致 OL 凋亡。 基于髓鞘再生的治疗有可能有助于保护轴突损伤和神经细胞死亡。 视网膜神经节细胞（RGC）神经元，减少慢性视力丧失，也许有助于恢复丧失的视力。 该项目的长期目标是确定参与 OL 分化和成熟的分子途径 可用于促进脱髓鞘视神经的髓鞘再生。为此，PI 已将其用于促进脱髓鞘视神经的髓鞘再生。 开发了为 RGC 和 OL 生产的人类多能干细胞 (hPSC)，并进行了单细胞 RNA- 测序和初步高通量筛选 (HTS) 来鉴定 OL 分化和 根据这些初步数据，PI 建议：1) 扩大筛选工作以识别。 OL 成熟中涉及的其他化合物和途径，2) 检查转录因子和 从初步工作中确定了其在 OL 分化中的作用途径，以及 3) 确定并 系统地表征参与 OL 规范和成熟的 microRNA (miRNA)。 拟议的研究中，PI 将开发创新工具，例如 hPSC 报告基因，用于通过诱导型细胞分化 OL CRISPRi（干扰）和 CRISPRa（激活）系统，以及功能性体外 hOL/hRGC 共培养系统 在指导阶段，PI 将在 Don Zack 博士的实验室中评估 hRGC 髓鞘形成。 基因组编辑以生成 CRISPRi 和 CRISPRa 系，并在 Wilmer 的 HCS 设施中接受培训，其中 将进行小分​​子筛选，以探索 OL 成熟中涉及的信号通路。 第二年，在Jay Baraban博士（microRNA生物学专家）的指导下，进行了miRNA研究 OL 还将通过 Ingo Ruczinski（生物统计学）的培训来补充指导阶段。 合作者），他将提供 HTS 的路径分析和命中验证方面的帮助。 PI 的共同导师 Peter Calabresi 博士是脱髓鞘疾病领域的领先科学家，他将定期与以下人士会面： 在指导期间，建议并帮助他优先考虑基因和途径以进行进一步检查。 阶段，PI 将定期与他的顾问委员会会面，参加科学会议，并继续他的工作 PI 为拟议的研究和职业发展提供了理想的环境。 Zack 博士拥有成熟的 hPSC 实验室、最先进的 HCS 设施以及与知名机构的合作 这将有助于 PI 建立良好的合作、学习新技术并 在一个完善的学术机构建立一个独立的研究实验室。