Kinase Multitargeting for Glaucoma Neuroprotection

激酶多靶点治疗青光眼神经保护

• 批准号: 10675240
• 负责人: Derek Stuart Welsbie
• 金额: $ 8.62万
• 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/10675240
• 关键词: Affect; Axon; Biology; CRISPR therapeutics; Cell Death; Cell Survival; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Cytoprotection; Dependovirus; Development; Electrophysiology (science); Enhancers; Genes; Glaucoma; Glycogen Synthase Kinases; Guide RNA; Knock-out; Lead; Leucine Zippers; Mediator of activation protein; Methods; Modeling; Mus; Muscle Cells; Nerve Crush; Neurites; Neurodegenerative Disorders; Optic Nerve; Outcome; Pathway interactions; Patients; Phosphotransferases; Physiologic Intraocular Pressure; RNA Interference; Rattus; Retina; Retinal Ganglion Cells; Rodent Model; Role; Signal Transduction; Streptococcus pyogenes; Structure; Testing; Therapeutic; Virus; Visual; Work; axon injury; axonal degeneration; base; design; functional genomics; glycogen synthase kinase 3 beta; improved; in vivo; insight; neuroprotection; novel; optic nerve disorder; preservation; promoter; 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.

项目摘要 – 青光眼是一种神经退行性疾病，其中视网膜神经节有特定的丧失 目前的治疗方法集中在降低眼压（IOP）上，尽管这可能是可行的。 为了推进可以补充的神经保护策略。 降低眼压，我们一直在使用高眼压识别初级 RGC 中的潜在神经保护靶点 这项工作的第一次迭代使用 RNA 干扰，鉴定了双重功能。 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经过验证 从那时起，我们就完成了包括青光眼在内的啮齿动物模型的生物学研究。 基于规则间隔短回文重复 (CRISPR) 的筛选，以鉴定其基因 进一步增强了 DLK/LZK 敲击抑制所赋予的 RGC 保护 本次筛选中的热门新产品。 是糖原合酶激酶三β (GSK-3β)，强调了我们的不可知筛选方法的实用性， 多个研究小组之前发现，虽然 GSK-3β 确实在轴突损伤后在 RGC 中被激活，但 GSK- 然而，我们已经证明，在 DLK/LZK 通路的情况下，单独丢失 3β 并不会增加 RGC 存活率。 抑制，GSK-3β 损失确实会导致 RGC 存活率进一步增加，此外，我们发现了一个意想不到的结果。 神经突变性与抑制 DLK/LZK 和 GSK-3β 的协同作用，可实现强大的神经突保护。 该提案的中心假设是 DLK/LZK 和 GSK-3β 合作，可能是由于它们的 双重磷酸化肌细胞增强因子 2A (MEF2A) 的能力，导致体细胞和轴突变性 为了最大程度地保护神经，需要同时抑制 DLK、LZK 和 GSK-3β。 在体内测试这一假设并创建体内基因多靶点的通用方法，我们有 开发了一种新型腺相关病毒 (AAV)/CRISPR 载体，该载体使用了关于紧凑 H1 的新颖见解。 启动子，允许引导 RNA (gRNA) 和化脓性链球菌 Cas9 (SpCas9) 在单个 AAV 中传递 病毒，将克服治疗性基因编辑领域的主要障碍。 将 AAV/CRISPR 载体转入多靶点 DLK/LZK/GSK-3β，在原代 RGC 中验证它们，然后使用 由此产生的细胞探索 MEF2A 作为 GSK-3β 和 DLK/LZK 信号传导关键汇聚点的作用。 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 结构/功能以及多靶点是否导致电生理活性的长期保存 小鼠视神经挤压模型中的 RGC 和轴突变性减少 最后，SA3 将使用更多。 治疗相关设计，其中 AAV/CRISPR 病毒提供所有 CRISPR 组件，以测试 假设 RGC 中的激酶多靶点可改善大鼠青光眼模型的视觉效果。 我们预计这一提议将带来针对轴突和体细胞联合的强有力的 RGC 神经保护策略 新型 AAV/CRISPR 疗法的保存和开发。