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Rescue of Cul3 haploinsufficiency phenotypes with CRISPR-mediated Cul3 activation

通过 CRISPR 介导的 Cul3 激活拯救 Cul3 单倍体不足表型

基本信息

批准号：
10527778
负责人：
LILIA M IAKOUCHEVA
金额：
$ 23.7万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10527778
关键词：
ASD patient Adult Affect Alleles Anatomy Antisense Oligonucleotides Architecture Axon Blood - brain barrier anatomy Brain Brain region CRISPR-mediated transcriptional activation CRISPR/Cas technology CUL3 gene Cell Line Clustered Regularly Interspaced Short Palindromic Repeats Cognitive deficits Copy Number Polymorphism Cytoskeleton Defect Degradation Pathway Dendrites Deubiquitination Development Developmental Delay Disorders Disease Enhancers Exhibits F-Actin Foundations Future Gene Expression Genes Genome engineering Goals Growth Guide RNA Human Hyperactive behavior Hyperactivity Intellectual functioning disability Intermediate Filaments Length Measures Mediating Microfilaments Modeling Molecular Mus Mutant Strains Mice Mutation Neonatal Neurodevelopmental Disorder Neurons Obesity Pathway interactions Patients Phenotype Population Control Promoter Regions Proteins Proteomics Qi RNA Splicing Risk Factors Seizures Short-Term Memory Signal Transduction Single Nucleotide Polymorphism Social Interaction Synapses System Temporal Lobe Epilepsy Testing Therapeutic Therapeutic Intervention Time Transcription Coactivator UBE3A gene Ubiquitination Up-Regulation Variant autism spectrum disorder behavioral phenotyping brain magnetic resonance imaging cullin-3 de novo mutation dosage dravet syndrome exome exome sequencing genetic variant genome-wide in vivo induced pluripotent stem cell interest lateral ventricle meetings molecular phenotype mouse model multi-electrode arrays neurogenesis neuronal growth nuclease postnatal development promoter protein expression small molecule social deficits spatiotemporal therapeutic target therapy development transcriptomics treatment strategy ubiquitin ligase

项目摘要

SUMMARY Rare and de novo single nucleotide variants (SNVs) and copy number variants (CNVs) are major risk factors for Neurodevelopmental Disorders (NDDs). The majority of NDD-associated SNVs affect a single allele of a gene, leading to haploinsufficiency. Correcting haploinsufficiency by increasing the expression level of the deficient allele could provide an attractive strategy for NDDs treatment. A variant of CRISPR, CRISPRa (CRISPR activation), offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters or enhancers. Here, we are proposing to apply CRISPRa to upregulate the levels of Cul3 ubiquitin ligase, a high-confidence gene for NDDs. We have recently generated a haploinsufficient Cul3 mouse model. Brain MRI found decreased volume of cortical regions starting from early postnatal development and persisting into adulthood. Spatiotemporal transcriptomic and proteomic profiling implicated cytoskeletal and synaptic defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity measured by multielectrode arrays (MEA) were reduced in Cul3 mutant mice. Cul3 mutant mice also exhibited hyperactive behavior, along with social and cognitive deficits. We hypothesize that upregulation of Cul3 dosage early in development with CRSIPRa will rescue some (or all) of the observed phenotypes, and will lay the basis for general therapeutic interventions in NDDs. The goal of this project is to demonstrate the feasibility of compensating for Cul3 haploinsufficiency by rebalancing neuronal, molecular, cellular and network activity phenotypes. We will achieve this goal through the following Specific Aims: (1) To evaluate rescue potential of cellular and molecular phenotypes in the Cul3+/- CRISPRa mice; (2) To evaluate rescue potential of brain architecture and cognitive deficits in Cul3+/- CRISPRa mice. Our study will represent an effective strategy for rebalancing Cul3 (and potentially other NDD genes) deficiency and correcting associated phenotypes.

总结