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Principal Investigator: John B Zuchero
Organization: STANFORD UNIVERSITY
Fiscal Year: 2021
Award: $432,982
Funding agency: National Institute of Neurological Disorders and Stroke
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 therapeutics. This treatment modality could structurally stabilize the 3’ UTRs of
myelin-related transcripts to increase their transport and translation in oligodendrocytes, thereby increasing
myelin production. Such a drug may be critical in the treatment of otherwise incurable demyelinating diseases.
Terms: <3' Untranslated Regions><3'UTR><Acute><Address><Anti-Sense Oligonucleotides><Antisense Agent><Antisense Oligonucleotides><Aran-Duchenne disease><Autologous><Axon><Basic Research><Basic Science><Binding><Biochemical><Biology><CNS Nervous System><Cancers><Carrier Proteins><Catalogs><Cell Body><Cell Differentiation><Cell Differentiation process><Cell membrane><Cells><Central Nervous System><Chemicals><Code><Coding System><Collaborations><Common Rat Strains><Communicable Diseases><Coupling><Cruveilhier disease><Cytoplasmic Membrane><DNA Molecular Biology><Data><Data Set><Dataset><Demyelinating Diseases><Demyelinating Disorders><Development><Disease><Disorder><Disseminated Sclerosis><Drugs><Dysfunction><Explosion><FISH Technic><FISH Technique><FISH analysis><FISH assay><Fluorescence In Situ Hybridization><Fluorescent in Situ Hybridization><Foundations><Functional disorder><Funding><Genetic Alteration><Genetic Change><Genetic defect><Genetics-Mutagenesis><Goals><Gold><Image><In Vitro><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Knowledge><Libraries><MAPseq><Maintenance><Malignant Neoplasms><Malignant Tumor><Mammalian Cell><Maps><Medication><Medicine><Messenger RNA><Methods><Microscopy><Modality><Molecular Biology><Molecular Interaction><Multiple Sclerosis><Multiplexed Analysis of Projections by Sequencing><Mutagenesis><Mutagenesis Molecular Biology><Mutate><Mutation><Myelin><Myelin Basic Proteins><Myelin Sheath><NGS Method><NGS system><Natural regeneration><Nerve Cells><Nerve Unit><Nervous System Diseases><Neural Cell><Neuraxis><Neurobiology><Neurocyte><Neurologic Disorders><Neurological Disorders><Neurons><Neurosciences Research><Non-Polyadenylated RNA><Oligodendrocytes><Oligodendrocytus><Oligodendroglia><Oligodendroglia Cell><Patients><Pharmaceutic Preparations><Pharmaceutical Preparations><Physiopathology><Plasma Membrane><Preventative strategy><Prevention strategy><Preventive strategy><Process><Production><Protocol><Protocols documentation><R-Series Research Projects><R01 Mechanism><R01 Program><RNA><RNA Gene Products><RNA Seq><RNA Sequences><RNA Transport><RNA sequencing><RNA vaccine><RNA-based vaccine><RNAseq><Rat><Rats Mammals><Rattus><Regeneration><Regulation><Reporter><Research><Research Grants><Research Project Grants><Research Projects><Ribonucleic Acid><Ribonucleic Acid Transport><Role><Specific qualifier value><Specified><Spinal Muscular Atrophy><Structure><Techniques><Technology><Testing><Therapeutic><Transcript><Translations><Transport Protein Gene><Transport Proteins><Transporter Protein><Variant><Variation><anti-sense agent><anti-sense oligo><antisense oligo><catalog><design><designing><developmental><dimethyl sulfate><dimethylsulfate><drug/agent><experiment><experimental research><experimental study><gain of function><genome mutation><global gene expression><global transcription profile><imaging><in vivo><insight><insular sclerosis><leukodystrophy><mRNA><mRNA Expression><mRNA vaccine><mRNA-based vaccine><malignancy><mouse model><murine model><mutant><myelination><neoplasm/cancer><nervous system disorder><neurobiological><neurological disease><neuronal><new drug target><new drug treatments><new druggable target><new drugs><new pharmacotherapy target><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutic target><new therapeutics><new therapy><new therapy approaches><new therapy target><next gen sequencing><next generation sequencing><next generation therapeutics><nextgen sequencing><novel><novel drug target><novel drug treatments><novel druggable target><novel drugs><novel pharmacotherapy target><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutic target><novel therapeutics><novel therapy><novel therapy approach><novel therapy target><oligodendrocyte precursor><pathophysiology><plasmalemma><programs><regenerate><regenerative><remyelination><single molecule><social role><structural biology><substantia alba><success><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><therapeutic target><transcriptome><transcriptome sequencing><white matter>