Axon Regeneration-Associated Gene Enhancers Promoting Successful CNS Nerve Regeneration

总结 该建议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。虽然基因治疗已经成为一种有前途的 尽管基因治疗是治疗CNS疾病和损伤的一种新方法，但目前这一代基因治疗载体存在着 毒性与促进治疗基因的强但不受调节的表达的调节序列有关。 此外，由于再生相关基因是暂时调节的，神经损伤的治疗 预计需要更微妙的基因表达控制比目前的治疗设计，以取代 单基因疾病中的缺陷基因因此，需要驱动治疗性基因转移的调控元件。 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， 调节经历轴突再生的神经元中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CNS轴突相关的再生活性基因调控序列 在斑马鱼中。斑马鱼模型提供了优于哺乳动物模型的明显优势，因为与哺乳动物不同， CNS神经损伤诱导支持轴突再生和功能恢复的基因表达的变化。 在斑马鱼中发现的增强子元件在哺乳动物中显示出类似的活性， 确立了习因此，斑马鱼提供了一个很好的体内模型，用于功能性评估再生特异性 增强剂。我们的长期目标研究目标是开发一种基因治疗方案，旨在促进 人类患者成功的视神经再生。拟议的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，形成长距离物理的远端序列 与再生相关基因启动子区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。目的1是鉴定增强子 靶向关键的再生相关转录因子，使用染色质构象捕获方法。 目的2是分离能够调节再生相关基因表达的增强子元件， 在斑马鱼中使用体内转基因报告基因测定验证推定的增强子序列。成功 这项工作的完成有望揭示精确调控时间基因的基因增强子。 在CNS神经再生过程中表达，从而有助于成年神经元的重编程， 再生轴突生长。这些结果是重要的和创新的，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 再生这一信息预计将对下一代的发展产生积极的影响， 基因表达载体需要推进中枢神经系统神经再生的研究和基因治疗。

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.