Kinase Multitargeting for Glaucoma Neuroprotection

激酶多靶点治疗青光眼神经保护

基本信息

批准号：
9764369
负责人：
Derek Stuart Welsbie
金额：
$ 39.38万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9764369
关键词：
Ablation Affect Animals Axon Biological Assay Biology Blindness Breeding CRISPR screen Cell Death Cell Survival Cells Cessation of life Clustered Regularly Interspaced Short Palindromic Repeats Complement Cytoprotection DNA Polymerase II DNA Polymerase III Dependovirus Development Drug Targeting Electrophysiology (science)Electroretinography Enhancers Evaluation Gene Combinations Gene Targeting Genes Genetic Genetic Transcription Genome Glaucoma Glycogen Synthase Kinases Guide RNA Health Promotion Histological Techniques Knock-out Label Lead Leucine Zippers Mechanics Mediator of activation protein Methods Modeling Mus Muscle Cells N-terminal Nature Nerve Crush Neurites Neurodegenerative Disorders Neuroprotective Agents Ocular Hypertension Optic Nerve Optical Coherence Tomography Outcome Pathway interactions Patients Pattern Phosphorylation Phosphotransferases Physiologic Intraocular Pressure Play Proteins RNA Interference RNA interference screen Rattus Retina Retinal Retinal Ganglion Cells Rodent Model Role SOX11 gene Signal Transduction Site Specificity Streptococcus pyogenes Structure System Testing Therapeutic Venous Virus Visual Work adeno-associated viral vector axon injury axonal degeneration base design disability fluorescence imaging functional genomics glycogen synthase kinase 3 beta improved in vivo insight interest jun Oncogene knockout gene neuronal survival neuroprotection novel optic nerve disorder preservation pressure prevent promoter response restriction enzyme retinal ganglion cell degeneration screening synergism therapeutic gene vector

项目摘要

PROJECT SUMMARY – Glaucoma is a neurodegenerative disease in which there is specific loss of retinal ganglion cells (RGCs). Current therapies center around lowering intraocular pressure (IOP) although this can be challenging in some patients. In order to advance towards a neuroprotective strategy that could complement IOP-lowering, we have been identifying potential neuroprotective targets in primary RGCs using high- throughput functional genomic screening. The first iteration of this work, using RNA interference, identified dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) as key mediators of RGC cell death and validated the biology in rodent models of optic neuropathy, including glaucoma. Since then, we have completed a clustered regularly-interspaced short palindromic repeat (CRISPR)-based screen in order to identify genes whose knockout further potentiates the RGC protection conferred by DLK/LZK inhibition. The top new hit in this screen was glycogen synthase kinase three beta (GSK-3β). Highlighting the utility of our agnostic screening approach, multiple groups have previously found that while GSK-3β is indeed activated in RGCs after axonal injury, GSK- 3β loss alone does not increase RGC survival. We have shown however, in the setting of DLK/LZK pathway inhibition, GSK-3β loss does lead to a further increase in RGC survival. Moreover, we found an unexpected synergy in neurite degeneration with inhibition of DLK/LZK and GSK-3β leading to robust neurite protection. The central hypothesis of this proposal is that DLK/LZK and GSK-3β cooperate, potentially as a result of their ability to dually phosphorylate myocyte enhancer factor 2A (MEF2A), to cause somal and axonal degeneration and that simultaneous inhibition of DLK, LZK and GSK-3β is required for maximal neuroprotection. In order to test this hypothesis in vivo and to create a generalizable method for gene multitargeting in vivo, we have developed a novel adeno-associated virus (AAV)/CRISPR vector. This uses a novel insight about the compact H1 promoter which allows both guide RNA (gRNA) and S. pyogenes Cas9 (SpCas9) to be delivered in a single AAV virus, overcoming a major hurdle in the field of therapeutic gene editing. Specific aim 1 (SA1) will develop AAV/CRISPR vectors to multitarget DLK/LZK/GSK-3β, validate them in primary RGCs and then use the resulting cells to explore the role of MEF2A as a key convergence point of GSK-3β and DLK/LZK signaling. SA2 will use AAV/CRISPR vectors in vivo to test whether DLK/LZK/GSK-3β inhibition affects normal retinal structure/function and whether multitargeting leads to long-term preservation of electrophysiologically-active RGCs and decreased axon degeneration in the mouse optic nerve crush model. Finally, SA3 will use a more therapeutically-relevant design, in which the AAV/CRISPR virus delivers all of the CRISPR components, to test the hypothesis that kinase multitargeting in RGCs improves visual outcomes in a rat glaucoma model. Together, we anticipate this proposal will lead to a robust RGC neuroprotective strategy for combined axonal and somal preservation and the development of a novel AAV/CRISPR therapeutic.

项目摘要 - 青光眼是一种神经退行性疾病，其中残留神经节的特异性丧失细胞（RGC）。当前疗法围绕降低眼内压（IOP）中心，尽管这可以是在某些患者中具有挑战性。为了迈向可以完成的神经保护策略降低IOP，我们一直在使用高级RGC中确定潜在的神经保护靶标吞吐量功能基因组筛选。这项工作的第一次迭代，使用RNA干扰确定了双重亮氨酸拉链激酶（DLK）和亮氨酸拉链激酶（LZK）作为RGC细胞死亡的关键介体并经过验证包括青光眼在内的视神经病变啮齿动物模型中的生物学。从那以后，我们完成了一个集群通常相互间隔的短圆锥体重复（基于CRISPR）的屏幕，以识别其基因 DLK/LZK抑制作用的RGC保护进一步敲除潜力。这个屏幕中的最高新打击是糖原合酶激酶三个β（GSK-3β）。强调我们不可知论筛选方法的实用性，以前，多组发现，虽然GSK-3β确实在轴突损伤后的RGC中被激活，但GSK- 仅3β损失不会增加RGC的存活。但是，我们已经显示在DLK/LZK途径的设置中抑制作用，GSK-3β确实会导致RGC存活进一步增加。而且，我们发现了一个意外的神经退行性的协同作用，抑制DLK/LZK和GSK-3β，从而实现了稳健的神经模型保护。该提议的核心假设是DLK/LZK和GSK-3β的合作，可能是由于它们双重磷酸化肌细胞增强子因子2a（MEF2A）的能力，导致索马和轴突变性对于最大的神经保护作用，需要对DLK，LZK和GSK-3β进行简单的抑制作用。为了在体内检验此假设，并为在体内创建一种可推广的基因多核方法的方法，我们有开发了一种新型与腺相关病毒（AAV）/CRISPR载体。这使用了关于紧凑型H1的新颖见解启动子允许在单个AAV中传递引导RNA（GRNA）和链球菌Cas9（SPCAS9）病毒，克服了治疗基因编辑领域的主要障碍。特定目标1（SA1）将发展 AAV/CRISPR向量向Muttitarget DLK/LZK/GSK-3β进行验证，然后在主要RGC中进行验证结果细胞探索MEF2A作为GSK-3β和DLK/LZK信号传导的关键收敛点的作用。 SA2 将在体内使用AAV/CRISPR向量来测试DLK/LZK/GSK-3β是否会影响正常残留结构/功能以及多野电是否会长期保存电生理学活性小鼠视神经挤压模型中的RGC和改善的轴突变性。最后，SA3将使用更多与治疗相关的设计，AAV/CRISPR病毒提供了所有CRISPR组件，以测试 RGC中的激酶多静脉曲器的假设改善了大鼠青光眼模型中的视觉结果。一起，我们预计该提案将导致轴突和索马的合并的RGC神经保护策略保存和新型AAV/CRISPR治疗的发展。