Enhancer-targeted correction of haploinsufficient autism risk genes

单倍体不足的自闭症风险基因的增强子靶向校正

基本信息

批准号：
10686119
负责人：
George Tsun-Te Chen
金额：
$ 7.38万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10686119
关键词：
3-Dimensional ASH1L gene ATAC-seq Affect Alleles Architecture Brain CRISPR-mediated transcriptional activation Cell Differentiation process Cell Maturation Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Development Disease Elements Enhancers Ethnic Origin Family Gene Activation Gene Combinations Gene Expression Genes Genetic Genetic Enhancer Element Genetic Risk Genetic Transcription Heritability Hi-C Human Image Immunofluorescence Immunologic In Vitro Individual KDM5B gene Maps Messenger RNA Methods Modeling Molecular Morphology Mutation Neuronal Differentiation Neurons Organoids Pathogenicity Pathway interactions Phenotype Physiological Play Prosencephalon Proteins Public Health Race Regulator Genes Regulatory Element Risk Risk Taking Role Statistical Study Synthetic Genes System Technology Testing Therapeutic Therapeutic Intervention Time Tissues Visualization Work autism spectrum disorder brain based cell type cohort de novo mutation disorder risk fetal gene correction genetic testing genome editing genome sequencing genome-wide improved in vivo induced pluripotent stem cell insight mutant nerve stem cell neurobiological mechanism neurodevelopment neurogenesis neuron development novel therapeutics overexpression predictive modeling response restoration risk variant single cell sequencing single-cell RNA sequencing socioeconomics spatiotemporal stem cells three dimensional cell culture

项目摘要

Project Summary/Abstract Autism Spectrum Disorder (ASD) is a highly heterogeneous and highly heritable disease with complex genetic contributions, and yet about 20% of genetic risk is imparted by de novo, or newly arising, mutations of major effect. The majority of mutations in these genes are either known to or predicted to lead to truncated mRNA and protein products, strongly indicating likely haploinsufficiency, in which a single copy of a functional gene is not sufficient for normal brain development or function. I propose a carefully staged approach to correct the effects of these mutations by activating expression from the non-mutant copy to restore gene expression to normal levels. I will leverage advances in gene editing technology - using CRISPR-A to target enhancer regions of high confidence ASD genes in stem cell-based 3D cortical spheroids (hCS) in vitro, which have been shown to recapitulate many features of in vivo cortex development. CRISPR-A has the significant advantages of leveraging endogenous gene expression regulatory elements without editing the genome or relying on artificial gene constructs for overexpression. I also leverage previous work where we have created high-confidence genome-wide enhancer maps that connect these transcriptional activating regions with their cognate genes. I propose to: i) functionally validate putative enhancer sequences for 12 high confidence ASD genes, ii) test selected enhancers in hCS to characterize their effect on neuronal differentiation, and iii) characterize changes in developmental and morphological phenotypes in hCS carrying ASD risk mutations compared to wildtype hCS, and determine the ability of CRISPR-A to rescue gene expression and restore wildtype development in mutant hCS. I will utilize diverse technologies, including single cell sequencing and CLARITY imaging, to comprehensively visualize the development of neuronal subtypes in cortical spheroids. Results from this study may not only provide a proof of principle for gene activation as a therapeutic intervention but will substantially enhance our understanding of the effects of haploinsufficiency in ASD genes and the neurobiological mechanisms whereby they impact neuronal development.

项目摘要/摘要自闭症谱系障碍（ASD）是一种高度异质且高度可遗传的疾病，具有复杂的遗传贡献，但大约有20％影响。这些基因中的大多数突变已知或预测会导致mRNA截断和蛋白质产物强烈表明可能的单倍不足，其中功能基因的单个副本不是足以正常的大脑发育或功能。我提出了一种经过精心上演的方法来纠正效果这些突变通过激活从非突出副本的表达以将基因表达恢复到正常水平。我将利用基因编辑技术的进步 - 使用CRISPR -A靶向较高的增强剂区域在体外，基于干细胞的3D皮质球体（HC）中的ASD基因的置信度已显示为概括了体内皮层发育的许多特征。 CRISPR-A具有很大的优势利用内源基因表达调节元件，而无需编辑基因组或依赖人工过表达的基因构建体。我还利用了以前的工作，在我们创造了高信心的地方全基因组增强子图将这些转录激活区与其同源基因联系起来。我提议：i）在功能上验证了12个高置信度ASD基因的假定增强子序列，ii）测试 HC中选定的增强剂以表征其对神经元分化的影响，ii）表征了变化与WildType HCS相比并确定CRISPR-A挽救基因表达并恢复突变体中野生型的能力 HCS。我将利用各种技术，包括单细胞测序和清晰度成像，全面地可视化皮质球体中神经元亚型的发展。这项研究的结果可能不仅可以提供基因激活作为治疗干预的原理证明，而且可以实质上增强我们对ASD基因和神经生物学的单倍弥补的影响的理解它们会影响神经元发展的机制。