Kinase Multitargeting for Glaucoma Neuroprotection

激酶多靶点治疗青光眼神经保护

• 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
• 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

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.

项目总结-青光眼是一种神经退行性疾病，其中存在视网膜神经节的特定损失 细胞（RGC）。目前的治疗围绕降低眼内压（IOP），尽管这可能是不必要的。 对一些患者来说是个挑战。为了发展一种神经保护策略， 为了降低IOP，我们一直在使用高浓度的药物来确定原发性RGC中的潜在神经保护靶点。 通量功能基因组筛选。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细胞死亡的关键介质，并验证 啮齿类动物视神经病变（包括青光眼）模型中的生物学。从那时起，我们已经完成了一个集群 基于规则间隔短回文重复序列（CRISPR）的筛选，以鉴定 敲除进一步增强了DLK/LZK抑制所赋予的RGC保护。最新的热门影片 糖原合成酶激酶3 β（GSK-3β）。强调我们不可知筛选方法的效用， 多个研究小组先前发现，虽然GSK-3β在轴突损伤后确实在RGC中被激活，但GSK-3β在轴突损伤后确实在RGC中被激活。 单独的3β丢失不会增加RGC存活。然而，我们已经表明，在DLK/LZK通路的背景下， 抑制，GSK-3β损失确实导致RGC存活的进一步增加。此外，我们发现了一个意想不到的 神经突变性与DLK/LZK和GSK-3β抑制的协同作用导致强大的神经突保护。 该提议的中心假设是DLK/LZK和GSK-3β合作，可能是由于它们的 能够双重磷酸化肌细胞增强因子2A（MEF 2A），导致体细胞和轴突变性 同时抑制DLK、LZK和GSK-3β是最大的神经保护作用所必需的。为了 为了在体内验证这一假设，并建立一种可推广的体内基因多靶向方法，我们 开发了一种新型腺相关病毒（腺相关病毒）/CRISPR载体。这使用了一个关于紧凑型H1的新见解 启动子，其允许向导RNA（gRNA）和S.化脓性链球菌Cas9（SpCas 9）在单个AAV中递送 病毒，克服了治疗性基因编辑领域的主要障碍。具体目标1（SA 1）将制定 AAV/CRISPR载体多靶向DLK/LZK/GSK-3β，在原代RGC中验证它们，然后使用 结果显示，MEF 2A是GSK-3β和DLK/LZK信号传导的关键汇聚点。SA2 将在体内使用AAV/CRISPR载体来测试DLK/LZK/GSK-3β抑制是否影响正常视网膜病变。 结构/功能以及多靶向是否导致电生理活性的长期保存 在小鼠视神经挤压模型中RGCs和轴突变性减少。最后，SA 3将使用更多 治疗相关的设计，其中AAV/CRISPR病毒递送所有CRISPR组分，以测试 RGCs中的激酶多靶向作用改善大鼠青光眼模型的视觉结果的假设。我们一起努力， 我们预计这一建议将导致一个强大的RGC神经保护策略，联合轴突和体细胞， 保存和开发新的AAV/CRISPR治疗剂。