PROJECT SUMMARY Recent successes in RNA medicine, including the first-in-class Spinraza treatment for spinal muscular atrophy, demonstrate RNA gain-of-function as a novel therapeutic modality for previously intractable neurological disease and raise the prospect of similar treatments for demyelinating diseases. Such treatments would target oligodendrocytes – the myelinating cells of the central nervous system (CNS) – for a novel and specific strategy for preventative or regenerative RNA medicine. To create myelin, oligodendrocytes extend cell projections that encircle adjacent axons. These concentrically wrapped layers of cell membrane undergo a process called compaction to generate mature myelin. Critical to this process is the localization of a subset of the oligodendrocyte transcriptome to the nascent myelin sheath for local translation. By more than an order of magnitude, the myelin basic protein (MBP) mRNA is the most highly abundant and most highly transported protein-coding transcript in oligodendrocytes, and the drug-induced regulation of its expression, processing, and transport could potentially augment myelination by these cells. Unfortunately, testing the viability of such a strategy is not currently possible due to a lack of understanding of the RNA molecular and structural biology that underlies MBP mRNA transport in oligodendrocytes. In partnership with oligodendrocyte biology collaborators, we have recently begun to address this knowledge gap. We have applied newly invented chemical probing and RNA sequencing technologies to reveal a previously unappreciated repertoire of secondary structures in the MBP 3’ untranslated region (3’ UTR, a region that is known to be necessary for MBP mRNA transport) as well as a catalog of hundreds of other highly transported oligodendrocyte mRNAs. These data suggest features that may be targeted or mimicked with antisense oligonucleotides (ASOs) to modulate MBP mRNA function but need to be rigorously tested. Here, we propose to complete this exploratory research by (1) testing the functional importance of MBP 3’ UTR secondary structures with in-cell mutate-rescue experiments recently invented by our lab and validating these structure-transport relationships through targeted structure perturbation or stabilization facilitated by anti-sense oligonucleotides, and (2) designing a minimal transport-inducing 3’ UTR using insights from high-throughput structure determination and structure-function characterization of all highly transported transcripts in oligodendrocytes. We will evaluate success in both aims through multiple orthogonal methods, including next-generation sequencing, biochemical structure determination, and quantitative single-molecule RNA imaging that we have collaboratively developed for the study of oligodendrocyte projections. The proposed basic science research establishes a previously missing RNA structural biology foundation needed for the design and testing of Spinraza-like ASO ther...