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

CRSIPR 筛选视网膜神经节细胞存活和轴突再生的新型调节因子

基本信息

批准号：
10402334
负责人：
ZHIGANG HE
金额：
$ 51.77万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10402334
关键词：
Address Adult Affect Axon Brain Cell Survival Clustered Regularly Interspaced Short Palindromic Repeats Development Down-Regulation Failure Gene Expression Genes Glaucoma Growth Guide RNA Individual Injury Intervention Label Lead Libraries Mediating Mediator of activation protein Methods Modeling Mus Mutation Natural regeneration Nerve Crush Neurodegenerative Disorders Optic Nerve PTEN gene Phase Process Protocols documentation Regulation Retina Retinal Ganglion Cells SOX11 gene Survivors Technology Testing Time Traumatic injury Viral Markers Vitreous body structure Work anterior chamber axon injury axon regeneration base cell regeneration cell type clinically relevant design genetic analysis genetic manipulation improved insight knock-down knockout gene loss of function neuron loss neuronal survival neuroprotection novel novel strategies programs promoter repaired resilience screening single-cell RNA sequencing transcription factor virtual

项目摘要

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.

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的失败轴突再生。横断所有视网膜神经节细胞（RGC）轴突的视神经挤压（ONC）通常是用于建模该过程，并寻求保护RGC并促进再生的干预措施。下列的在小鼠中，约80％的RGC在2周内死亡，几乎没有幸存者再生轴突。我们和其他人使用ONC来确定导致生存增加，再生增加的干预措施或两者兼而有之。但是，这些治疗仅是部分有效的。例如，PTEN删除增加RGC 生存仅为两倍，> 45种RGC类型，只有少数（alpha-rgc）扩展了轴突。另外，再生轴突的增长率很慢，最重要的是，再生轴突很少达到其大脑中的目标。因此，重要的是要确定生存的额外和改进的启动子和再生。我们将通过使用公正的功能丢失屏幕来应对这一挑战 CRISPR（群集定期间隔短的短粒子重复序列）。根据我们以前的工作和其他显示敲低调节几个转录因子可以提高生存率，再生或两者都为1629个转录因子基因生成了一个基于AAV2的SGRNA库。优化将SGRNA和CAS9传递到RGC的方法；并验证了我们识别调节基因的能力生存和再生。在拟议的研究中，我们将筛选整个图书馆，以找到神经保护和再生所需的程序。对于选定的积极命中，我们将确定RGC 受保护和/或在单个基因敲除之后受到保护和/或经历轴突再生的亚型。最后，作为一个在临床上相关的环境中测试确定的候选人的第一步，我们将以强劲的方式选择三个GRNA 神经保护作用并测试其在广泛使用的青光眼模型中保护RGC的能力。我们期望这些研究将提供洞察力，使新的神经保护和再生发展 - 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。