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在于神经元死亡和受损神经元的功能衰竭。 轴突再生视神经压碎（ONC），其横切所有视网膜神经节细胞（RGC）轴突，通常是视网膜神经节细胞（RGC）的一部分。 用于模拟这一过程，并寻求保护RGC和促进再生的干预措施。以下 在小鼠中，约80%的RGC在2周内死亡，并且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 或两者然而，这些治疗方法只是部分有效。例如，PTEN缺失增加RGC 存活率仅为两倍，且在>45种RGC类型中，仅少数（α-RGC）延伸轴突。另夕h 再生轴突的生长速度很慢，最重要的是，再生轴突很少能到达它们的 大脑中的目标。因此，重要的是鉴定另外的和改进的存活促进剂， 再生我们将通过使用以下方法进行无偏见的功能丧失筛查来应对这一挑战 CRISPR（英语：CRISPR）是一种规则间隔的短回文重复序列。基于我们以前的工作， 其他研究表明，敲低调节几种转录因子可以提高存活率，再生， 或两者，我们已经产生了1629个转录因子基因的基于AAV 2的sgRNA文库;优化 将sgRNA和Cas9递送到RGCs的方法;并验证了我们鉴定调节RGCs的基因的能力。 生存和再生。在拟议的研究中，我们将筛选整个文库，以寻找新的阻遏物。 神经保护和再生所需的程序。对于选定的阳性结果，我们将确定研资局 在单个基因敲除后受保护和/或经历轴突再生的亚型。最后，作为 为了在临床相关环境中测试所鉴定的候选物，我们将选择三种具有鲁棒性的gRNA。 神经保护作用，并测试它们在广泛使用的青光眼模型中保护RGC的能力。我们预计 这些研究将提供见解，使新的神经保护和再生的发展- 促进创伤性损伤和青光眼以及其他神经退行性疾病的策略。

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.