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：
10672996
负责人：
LILIA M IAKOUCHEVA
金额：
$ 19.75万
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/10672996
关键词：
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 Compensation Convulsants Copy Number Polymorphism Cytoskeletal Filaments 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 Protein Truncation 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.

概括罕见和从头的单核苷酸变异（SNV）和拷贝数变异（CNV）是主要的危险因素神经发育障碍（NDD）。大多数 NDD 相关 SNV 影响基因的单个等位基因，导致单倍体不足。通过增加缺陷的表达水平来纠正单倍体不足等位基因可以为 NDD 治疗提供有吸引力的策略。 CRISPR 的一种变体，CRISPRa（CRISPR 激活），提供了通过直接靶向内源基因来调节内源基因表达的可能性启动子或增强子。在这里，我们建议应用CRISPRa来上调Cul3泛素的水平连接酶，NDD 的高置信度基因。我们最近生成了一个单倍剂量不足的 Cul3 小鼠模型。脑部 MRI 发现，皮质区域的体积从出生后早期发育开始就开始减少，并持续存在进入成年期。涉及细胞骨架和突触的时空转录组和蛋白质组分析缺陷是 Cul3 功能影响的关键驱动因素。具体来说，树突生长、丝状肌动蛋白斑点和在 Cul3 突变小鼠中，通过多电极阵列 (MEA) 测量的自发网络活动减少。铜3 突变小鼠还表现出过度活跃的行为，以及社交和认知缺陷。我们假设在开发早期使用 CRSIPRa 上调 Cul3 剂量将挽救一些（或全部）观察到的情况表型，并将为 NDD 的一般治疗干预奠定基础。该项目的目标是为了证明通过重新平衡神经元、分子、细胞和网络活动表型。我们将通过以下具体目标来实现这一目标：（1）评估 Cul3+/- CRISPRa 小鼠细胞和分子表型的拯救潜力； (2) 评价拯救 Cul3+/- CRISPRa 小鼠大脑结构和认知缺陷的潜力。我们的研究将代表重新平衡 Cul3（以及可能的其他 NDD 基因）缺陷和纠正相关的有效策略表型。