Kinase Multitargeting for Glaucoma Neuroprotection

项目摘要-青光眼是一种神经退行性疾病，视网膜神经节有特殊的丢失。 细胞(RGC)。目前的治疗以降低眼压(IOP)为中心，尽管这可能是 在一些患者中具有挑战性。为了推进神经保护策略，可以补充 为了降低眼压，我们一直在使用高眼压技术在初级视网膜节细胞中识别潜在的神经保护靶点。 通过功能基因组筛选。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及其验证 包括青光眼在内的视神经病变啮齿动物模型的生物学。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)的筛查，以识别其基因 基因敲除进一步增强了DLK/LZK抑制对RGC的保护作用。这个屏幕上最新的热门歌曲 糖原合成酶激酶3β(GSK-3β)。突出了我们的不可知性筛选方法的实用性， 多个研究小组此前已发现，虽然轴突损伤后视网膜神经节细胞中确实存在GSK-3β的激活，但GSK-3 3仅β缺失并不能增加RGC的存活率。然而，我们已经证明，在DLK/LZK通路的设置中 抑制后，GSK-3β缺失确实会导致RGC存活率进一步增加。此外，我们发现了一个意想不到的 抑制DLK/LZK和GSK-3β在轴突退变中的协同作用导致强大的轴突保护。 这一提议的中心假设是DLK/LZK和GSK-3β合作，可能是由于他们的 双磷酸化肌细胞增强因子2A(MEF2A)导致胞体和轴突变性的能力 同时抑制DLK、LZK和GSK-3β才能发挥最大的神经保护作用。为了 在体内测试这一假设，并创建一种可推广的体内基因多靶向方法，我们有 构建了一种新型腺相关病毒(AAV)/CRISPR载体。这使用了对紧凑型H1的新见解 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，克服了治疗性基因编辑领域的一大障碍。将制定特定目标1(SA1) AAv/CRISPR载体到多靶点DLK/LZK/GSK-3β，在原代RGC中验证它们，然后使用 从而探索MEF2a作为GSK-3β和DLK/LZK信号的关键汇聚点的作用。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 结构/功能以及多靶点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，视网膜节细胞和减少轴突变性。最后，SA3将使用更多 与治疗相关的设计，其中AAV/CRISPR病毒提供所有CRISPR组件以进行测试 在大鼠青光眼模型中，视网膜节细胞中的激酶多靶向性改善了视觉结果的假说。一起， 我们预计这项提议将导致一个强大的RGC神经保护策略，用于轴突和索状体的联合保护 一种新型AAV/CRISPR治疗药物的保存和开发

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.