Axon Regeneration-Associated Gene Enhancers Promoting Successful CNS Nerve Regeneration

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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.