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Principal Investigator: Rocco George Gogliotti
Organization: LOYOLA UNIVERSITY CHICAGO
Fiscal Year: 2024
Award: $192,500
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
SUMMARY
An emerging theme in neurodevelopmental research is that both loss of function and overexpression of
the same pathogenic gene can result in autism-associated phenotypes. For many disorders, such precise
requirements for protein dosage have complicated what was once thought to be a linear path to gene therapy.
This challenge is epitomized by Rett syndrome, a monogenic neurodevelopmental disorder caused by loss of
function mutations in a methyl-reader known as Methyl-CpG Binding Protein 2 (MeCP2). On the surface, RTT
appears to be an ideal candidate for gene therapy; however, targeting MeCP2 itself with traditional gene
replacement strategies is complicated by a stringent requirement for protein dosage, whereby even a 1-fold
increase over neurotypical levels evokes adverse effects. The practical challenge created by these narrow
dosage requirements is that, not only does viral MeCP2 delivery need to be efficient across the entire human
brain, but each must cell receive roughly the same, relatively small amount. One endogenous mechanism used
to fine-tune MeCP2 expression is via microRNA (miRNA) regulation of its 3’untranslated region (UTR). As the
contribution of each miRNA to MeCP2 expression is modest by nature, we hypothesized that preventing the
binding of repressive-miRNAs would be a viable approach to increase MeCP2 dosage, yet remain within its
narrow safety margins. To test this hypothesis, we developed locked nucleic acid (LNA) site blocking (sb) ASOs
designed to outcompete mir-22, mir-132, and mir-483 for binding to the MeCP2 3’UTR. In support of our
hypothesis, increasing concentrations of each ASO in vitro resulted in increasing amounts of MeCP2 protein to
a point that plateaued at a 0.75 to 4-fold increase, depending on the miRNA site being blocked. We contend that
this approach is ideally suited for patients with common missense or late-truncating mutations, where some
function is preserved and where overexpression of the mutant protein is known to improve phenotypes in mice.
In patients, eight MeCP2 mutations are responsible for 70% of all RTT cases, and five of these are missense
and late-truncating, potentially suggesting a broad utility. In Aim 1, we will use fibroblast and iPSC-derived
neurons from RTT patients to determine the subpopulations where overexpression of the mutant protein
demonstrates efficacy. In Aim 2, we will use osmotic micropumps to deliver a dose-response of each sbASO to
Mecp2T158M/y mice and establish a therapeutic range for efficacy and adverse effects. The T158M mouse model
was chosen because transgenic overexpression of the mutant allele has already been shown to be effective at
improving phenotypes. In recent years, advancements in ASO chemistry have removed many of the long-
standing barriers preventing their clinical development. Here we propose that LNA-modified ASOs designed to
outcompete endogenous miRNA for MeCP2 regulation are a viable therapeutic approach for RTT patients with
missense or late-truncating mutations.
Terms: <3' Untranslated Regions><3'UTR><ASD><Adverse effects><Alleles><Allelomorphs><Antisense Agent><Antisense Oligonucleotides><Aran-Duchenne disease><Autism><Autistic Disorder><Binding><Binding Sites><Brain><Brain Nervous System><Candidate Disease Gene><Candidate Gene><Cell Body><Cell Line><CellLine><Cells><Cementation><Cerebroatrophic Hyperammonemia><Chemicals><Chemistry><Combining Site><Cruveilhier disease><DNA Therapy><Data><Development><Disease><Disorder><Dose><Early Infantile Autism><Encephalon><Fibroblasts><Foundations><Gene Expression><Gene Transfer Clinical><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Goals><Human><In Vitro><Individual><Induced pluripotent stem cell derived neurons><Infantile Autism><Kanner's Syndrome><Kinetics><MeCP-2 protein><MeCP2><MeCP2 Duplication Syndrome><MeCP2 protein><Messenger RNA><Methyl CpG Binding Protein 2><Methyl CpG binding protein MeCP2><Methyl-CpG binding protein 2><Methyl-CpG-Binding Protein 2><Methyl-DNA binding protein MECP2><Mice><Mice Mammals><Micro RNA><MicroRNAs><Modern Man><Modification><Molecular Interaction><Morphology><Murine><Mus><Mutation><Nature><Neurodevelopmental Disorder><Neurological Development Disorder><Neuron from iPSC><Neuron from induced pluripotent stem cells><Neurosciences><Osmosis><Outcome Measure><Pathology><Patients><Phenotype><Proteins><Pump><RNA Splicing><RNA-Binding Proteins><Reactive Site><Reader><Regulation><Reproducibility><Research><Rett Disorder><Rett Syndrome><Role><Safety><Seminal><Series><Site><Spinal Column><Spinal Muscular Atrophy><Spine><Splicing><Strains Cell Lines><Sugar Phosphates><Surface><Symptoms><Synapses><Synaptic><Syndrome><Testing><Therapeutic><Transgenic Organisms><Vertebral column><Viral><Viral Genes><antisense oligo><autism spectral disorder><autism spectrum disorder><autistic spectrum disorder><backbone><clinical development><cultured cell line><derepression><design><designing><developmental><dosage><effective therapy><effective treatment><experience><gene repair therapy><gene replacement><gene therapy><gene-based therapy><genetic therapy><genome mutation><genomic therapy><iPS neurons><iPSC derived-neurons><improved><in vivo><induced pluripotent stem cell neurons><locked nucleic acid><loss of function><loss of function mutation><mRNA><mRNA Expression><measurable outcome><methyl CpG-binding protein 2 duplication syndrome><miRNA><miRNAs><mouse model><murine model><mutant><neuroblastoma cell><neurodevelopmental disease><neurons derived from induced pluripotent stem cells><oligonucleotide delivery><outcome measurement><overexpress><overexpression><pathogenicity gene><patient subclass><patient subcluster><patient subgroups><patient subpopulations><patient subsets><patient subtypes><posttranscriptional><preservation><prevent><preventing><protein expression><response><restoration><safety assessment><social role><synapse><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><theories><tool><transgenic><treatment strategy><virulence gene><virulent gene>