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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：
9920148
负责人：
ZHIGANG HE
金额：
$ 53.37万
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/9920148
关键词：
Address Adult Affect Axon Brain Cell Survival Clustered Regularly Interspaced Short Palindromic Repeats Development Down-Regulation Expression Profiling 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功能衰竭轴突再生。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。下列的在小鼠 ONC 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 来确定可提高存活率和再生能力的干预措施或两者兼而有之。然而，这些治疗仅部分有效。例如，PTEN 缺失会增加 RGC 存活率仅提高两倍，并且在 > 45 种 RGC 类型中，只有少数（α-RGC）延长轴突。此外，再生轴突的生长速度缓慢，最重要的是，再生轴突很少达到其应有的水平。大脑中的目标。因此，识别额外的和改进的生存促进剂和再生。我们将通过使用以下方法进行公正的功能丧失筛查来应对这一挑战： CRISPR（成簇规则间隔短回文重复序列）。根据我们之前的工作和其他研究表明，几种转录因子的敲低调节可以提高存活率和再生能力或者两者兼而有之，我们已经为 1629 个转录因子基因生成了基于 AAV2 的 sgRNA 文库；优化的将 sgRNA 和 Cas9 递送至 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，我们将筛选整个库以寻找新的抑制子神经保护和再生所需的计划。对于选定的积极点击，我们将确定 RGC 在个体基因敲除后受到保护和/或经历轴突再生的亚型。最后，作为一个在临床相关环境中测试已识别候选者的第一步，我们将选择三种具有稳健性的 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见解，使新型神经保护和再生药物的开发成为可能。促进针对创伤性损伤和青光眼以及其他神经退行性疾病的策略。