Axon Regeneration-Associated Gene Enhancers Promoting Successful CNS Nerve Regeneration

摘要 该提案旨在鉴定能够调节时间动态基因表达的增强子元件 与成功的中枢神经系统神经再生有关。尽管基因治疗已成为一种有前途的治疗方法 治疗中枢神经系统疾病和损伤的方法，当前一代的基因治疗载体存在风险 毒性与促进治疗基因强烈但不受调节的表达的调节序列有关。 此外，由于再生相关基因受到暂时调节，神经损伤的治疗 预计与目前旨在替代的治疗方法相比，需要对基因表达进行更细致的控制 单基因疾病中的缺陷基因。因此，需要驱动治疗基因的调控元件 以特定上下文的方式表达。然而，我们对 DNA 序列的理解存在严重差距 调节经历轴突再生的神经元的时间动态基因表达。我们建议确定 并在功能上验证与成功的中枢神经系统轴突相关的再生活性基因调控序列 在斑马鱼中。斑马鱼模型比哺乳动物模型具有明显的优势，因为与哺乳动物不同， 中枢神经系统神经损伤会引起支持轴突再生和功能恢复的基因表达变化。 在斑马鱼中发现的增强子元件在哺乳动物中表现出类似的活性 成立。因此，斑马鱼提供了一个出色的体内模型，用于功能评估再生特异性 增强剂。我们的长期目标研究目标是开发一种基因治疗计划，旨在促进 人类患者的视神经成功再生。拟议 R03 项目的目标是确定 并在功能上验证足以促进暂时动态基因表达的基因调控序列 视神经再生期间。我们的中心假设是远端序列形成远程物理 与再生相关基因启动子区域的相互作用调节基因的时间特异性 表达。我们将通过追求以下具体目标来检验我们的假设。目标 1 是识别增强子 使用染色质构象捕获方法，针对关键的再生相关转录因子。 目标 2 是分离能够调节再生相关基因表达的增强子元件，以及 使用斑马鱼体内转基因报告基因测定验证推定的增强子序列。成功 拟议工作的完成预计将揭示精确调节时间基因的基因增强子 在中枢神经系统神经再生过程中表达，从而有助于成年神经元的重新编程 再生轴突生长。这些结果具有重大意义和创新性，因为它们将阐明增强剂 能够在经历轴突的 CNS 神经元中产生生理相关基因表达的元件 再生。这些信息预计将对下一代的发展产生积极影响。 推进中枢神经系统神经再生研究和基因治疗所需的基因表达载体。

SUMMARY This proposal aims to identify enhancer elements capable of regulating temporally dynamic gene expression associated with successful CNS nerve regeneration. Although gene therapy has emerged as a promising approach to treat CNS disorders and injuries, the current generation of gene therapy vectors carry a risk of toxicity linked to regulatory sequences that promote strong, but unregulated expression of therapeutic genes. Furthermore, because regeneration-associated genes are temporally regulated, the treatment of nerve injuries is predicted to require more nuanced control of gene expression than current treatments designed to replace faulty genes in monogenic diseases. Thus, there is a need for regulatory elements that drive therapeutic gene expression in a context-specific manner. However, there is a critical gap in our understanding of DNA sequences regulating temporally dynamic gene expression in neurons undergoing axon regeneration. We propose to identify and functionally validate regeneration-active gene regulatory sequences associated with successful CNS axon in zebrafish. Zebrafish models provide a distinct advantage over mammalian models because, unlike mammals, CNS nerve injury induces changes in gene expression that support axon regeneration and recovery of function. Enhancer elements discovered in zebrafish that show analogous activity in mammals has been previously established. Thus, zebrafish provide an excellent in vivo model for functionally evaluating regeneration-specific enhancers. Our long-term goal research goal is to develop a program of gene therapy designed to promote successful optic nerve regeneration in human patients. The objective of the proposed R03 project is to identify and functionally validate gene regulatory sequences sufficient for promoting temporally dynamic gene expression during optic nerve regeneration. Our central hypothesis is that distal sequences forming long range physical interactions with the promoter regions of regeneration-associated genes regulate temporal specificity of gene expression. We will test our hypothesis by pursuing the following specific aims. Aim 1 is to identify enhancers targeting key regeneration-associated transcription factors, using a chromatin conformation capture approach. Aim 2 is to isolate enhancer elements capable of regulating regeneration-associated gene expression, and validate putative enhancer sequences using in vivo transgenic reporter assays in zebrafish. Successful completion of the proposed work is expected to reveal gene enhancers that accurately regulate temporal gene expression during CNS nerve regeneration and thus contribute to the reprogramming of adult neurons for regenerative axon growth. These outcomes are significant and innovative because they will elucidate enhancer elements capable of producing physiologically relevant gene expression in CNS neurons undergoing axon regeneration. This information is expected to have positive impact on the development of the next generation of gene-expression vectors needed to advance research and gene therapy treatments for CNS nerve regeneration.