CRSIPR screening for novel regulators of retinal ganglion cell survival and axonal regeneration

摘要/项目摘要 包括青光眼在内的神经退行性疾病的特征是神经元死亡和受损失败。 轴突再生。视神经挤压(ONC)横断所有视网膜神经节细胞(RGC)轴突，通常是 用于模拟这一过程，并寻求保护RGC和促进再生的干预措施。跟随 在小鼠中，约80%的视网膜节细胞在2周内死亡，几乎没有幸存者再生轴突。 我们和其他人已经使用ONC来确定导致增加存活率、增加再生的干预措施 或者两者都有。然而，这些治疗方法只有部分效果。例如，PTEN缺失会增加RGC 只有两倍存活，而且在45个RGC类型中，只有少数(α-RGC)延伸轴突。此外， 再生轴突的生长速度很慢，最重要的是，再生轴突很少达到 大脑中的靶子。因此，重要的是要确定更多和更好的生存和 再生。我们将通过使用以下工具进行无偏见的功能丧失屏幕来应对这一挑战 CRISPR(成簇的规则间隔的短回文重复)。基于我们之前的工作和 另一些研究表明，几种转录因子的敲除调控可以改善存活、再生 或者两者兼而有之，我们已经为1629个转录因子基因生成了一个基于AAV2的sgRNA文库；优化 将sgRNAs和Cas9传递给RGC的方法；并验证了我们识别调控基因的能力 生存和再生。在这项拟议的研究中，我们将对整个图书馆进行筛选，以寻找新的抑制因子 神经保护和再生所需的计划。对于选定的肯定命中，我们将确定RGC 受保护的和/或在单个基因敲除后经历轴突再生的亚型。最后，作为一个 第一步是在临床相关的环境中测试确定的候选基因，我们将选择三个具有健壮性的gRNA 并在广泛使用的青光眼模型中测试它们保护视网膜节细胞的能力。我们预计 这些研究将提供洞察力，使开发新的神经保护和再生- 促进创伤性损伤和青光眼以及其他神经退行性疾病的战略。

Abstract/Project Summary Neurodegenerative diseases including glaucoma are characterized by neuronal death and failure of damaged axons to regenerate. Optic nerve crush (ONC), which transects all retinal ganglion cell (RGC) axons, is often used to model this process, and to seek interventions that protect RGCs and promote regeneration. Following ONC in mice, ~80% of the RGCs die within 2 weeks, and virtually none of the survivors regenerate axons. We and others have used ONC to identify interventions that lead to increased survival, increased regeneration or both. However, these treatments are only partially effective. For example, PTEN deletion increases RGC survival by only two-fold, and of >45 RGC types, only a few (alpha-RGCs) extend axons. Additionally, the growth rates of regenerating axons are slow and, most important, the regenerating axons rarely reach their targets in the brain. It is therefore important to identify additional and improved promoters of survival and regeneration. We will address this challenge by conducting an unbiased loss-of-function screen using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Based on previous work from us and others showing that knockdown-regulation of several transcription factors can improve survival, regeneration or both, we have generated an AAV2-based sgRNA library for 1629 transcription factor genes; optimized methods for delivering the sgRNAs and Cas9 to RGCs; and validated our ability to identify genes that regulate survival and regeneration. In the proposed study, we will screen the entire library to find novel repressors of programs required for neuroprotection and regeneration. For selected positive hits, we will identify RGC subtypes that are protected and/or undergo axon regeneration after individual gene knockout. Finally, as a first step to test identified candidates in a clinically relevant setting, we will choose three gRNAs with robust neuroprotective effects and test their ability to protect RGCs in a widely-used glaucoma model. We expect these studies will provide insights that will enable development of novel neuroprotective and regeneration- promoting strategies for traumatic injury and glaucoma as well as other neurodegenerative diseases.