Axon Regeneration-Associated Gene Enhancers Promoting Successful CNS Nerve Regeneration

轴突再生相关基因增强剂促进中枢神经系统神经再生成功

• 批准号: 10511016
• 负责人: AVA J UDVADIA
• 金额: $ 6.9万
• 依托单位: APPALACHIAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10511016
• 关键词: Acute; Address; Adult; Automobile Driving; Axon; Biological Assay; Central Nervous System Diseases; Chromatin; DNA Sequence; Data; Development; Disease; Distal; Elements; Enhancers; Gene Expression; Gene Expression Profile; Gene Transduction Agent; Generations; Genes; Genetic Enhancer Element; Goals; Human; Injury; JUN gene; Knowledge; Link; Mammals; Maps; Mendelian disorder; Mission; Modeling; National Institute of Neurological Disorders and Stroke; Natural regeneration; Nerve Degeneration; Nerve Regeneration; Neurodegenerative Disorders; Neurons; Optic Nerve Injuries; Outcome; Patients; Pattern; Physiological; Promoter Regions; Public Health; Publishing; Recovery of Function; Regulator Genes; Regulatory Element; Reporter Genes; Research; Risk; Safety; Screening procedure; Specificity; System; Testing; Time; Toxic effect; Transgenic Organisms; Viral Genes; Work; Zebrafish; axon growth; axon regeneration; cardiac regeneration; central nervous system injury; chromosome conformation capture; deletion analysis; digit regeneration; efficacy testing; experimental study; expression vector; gene therapy; in vivo; in vivo Model; innovation; mouse model; nerve injury; next generation; optic nerve regeneration; prevent; programs; promoter; regenerative; therapeutic gene; therapy design; transcription factor

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.

摘要 这项建议旨在确定能够调节时间动态基因表达的增强子元件 与成功的中枢神经再生有关。尽管基因疗法已经成为一种很有前途的 治疗中枢神经系统疾病和损伤的方法，当前一代基因治疗载体携带有 毒性与促进治疗基因强烈但不受调控的表达的调控序列有关。 此外，由于再生相关基因是暂时调节的，神经损伤的治疗 预计需要对基因表达进行更细微的控制，而不是目前旨在取代 单基因疾病中的缺陷基因。因此，需要调控元件来驱动治疗性基因。 以特定于上下文的方式表达。然而，在我们对DNA序列的理解上存在着严重的差距 调节轴突再生过程中神经元的时间动态基因表达。我们建议确定 并从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 在斑马鱼身上。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， 中枢神经系统损伤导致支持轴突再生和功能恢复的基因表达发生变化。 在斑马鱼中发现的在哺乳动物中表现出类似活性的增强子元素之前 已经成立了。因此，斑马鱼提供了一个很好的体内模型，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 人类患者成功的视神经再生。建议的R03项目的目标是确定 并在功能上验证足以促进时间动态基因表达的基因调控序列 在视神经再生过程中。我们的中心假设是形成远程物理序列的远端序列 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 表情。我们将通过追求以下具体目标来检验我们的假设。目标1是确定增强剂 以关键的再生相关转录因子为靶点，使用染色质构象捕捉方法。 目的2是分离能够调节再生相关基因表达的增强子元件，以及 在斑马鱼中使用体内转基因报告分析验证假定的增强子序列。成功 拟议工作的完成有望揭示准确调控时间基因的基因增强剂 在CNS神经再生过程中的表达，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子 在经历轴突的中枢神经系统神经元中能够产生生理相关基因表达的元件 再生。预计这一信息将对下一代网络的发展产生积极影响 需要基因表达载体来推进中枢神经再生的研究和基因治疗。