Enhancer-targeted correction of haploinsufficient autism risk genes

项目概要/摘要 自闭症谱系障碍（ASD）是一种高度异质性和高度遗传性的疾病，具有复杂的遗传学特征。 然而，大约20%的遗传风险是由新生的，或新出现的，主要基因突变引起的。 效果这些基因中的大多数突变已知或预测会导致截短的mRNA， 蛋白质产物，强烈表明可能的单倍不足，其中功能基因的单拷贝不是 足够正常的大脑发育或功能。我提出了一个谨慎的阶段性方法来纠正影响 通过激活非突变拷贝的表达，使基因表达恢复正常， 程度.我将利用基因编辑技术的进步-使用CRISPR-A靶向高表达的增强子区域， 在体外基于干细胞的3D皮质球状体（hCS）中置信ASD基因，已显示其 概括了体内皮质发育的许多特征。CRISPR-A具有以下显著优势： 利用内源性基因表达调控元件，而不编辑基因组或依赖人工 用于过表达的基因构建体。我还利用以前的工作，我们已经创造了高信心， 全基因组增强子图谱将这些转录激活区域与它们的同源基因连接起来。我 建议：i）功能验证12个高置信度ASD基因的推定增强子序列，ii）测试 在hCS中选择增强子以表征它们对神经元分化的作用，和iii）表征变化 与野生型hCS相比，在携带ASD风险突变的hCS的发育和形态表型中， 并确定CRISPR-A在突变体中拯救基因表达和恢复野生型发育的能力。 hCS。我将利用各种技术，包括单细胞测序和生物成像， 全面可视化皮质球状体中神经元亚型的发育。本研究结果 不仅可以提供基因激活作为治疗干预的原理证明， 加强我们对ASD基因单倍不足的影响和神经生物学的理解， 影响神经元发育的机制。

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.