Defining Informative Loci to Enable Mechanistic Insight and Treatment of Chromatinopathies

项目摘要 近几十年来，基因组分析已经阐明了数百种疾病的病因 综合征型和非综合征型智力残疾（ID）。然而，这些知识并不容易 转化为对患者的治疗，部分原因是治疗开发策略只关注单一的 疾病许多患有ID的患者在编码染色质组织蛋白的基因中存在突变。 cohesin是一种关键的染色质调节蛋白，其突变导致以下患者的临床表现重叠： 在其他染色质调节复合物中有突变，包括BAF/SWI-SNF和p160/NCOA。 长期目标是确定治疗方法，以改善儿童的神经认知结果， 染色质调节障碍。本R21的总体目标是确定共同的信息目标 在实验上可接近的和患者来源的细胞中， 细胞模型中心假设是，粘附素、BAF/SWI-SNF和p160/NCOA复合物中的突变 导致相似的临床特征，因为它们破坏了一组重叠的靶基因。基本原理是 鉴定常见的、可重复的染色质破坏将使突破性的工作能够快速筛选 在实验室环境中使用药物，并在治疗前在患者细胞中确认精确治疗。 这一中心假设将通过以下两个具体目标进行检验：1）确定 在具有粘附素、BAF/SWI-SNF和p160/NCOA杂合突变的HCT 116细胞中的染色质破坏 复杂基因;和2）鉴定患者来源的淋巴母细胞样瘤中染色质破坏的共同区域 细胞对于第一个目标，CRISPR-Cas9将用于为每个基因组创建杂合的功能丧失突变。 六个染色质调节基因的基因组。RNA-seq和ATAC-seq将用于鉴定 由此产生的改变基因表达和染色质可及性的模式。在第二个目标下， 研究人员收集了大量的异质性患者淋巴母细胞样细胞系，这些细胞系在三个 染色质调控基因将被用来确定改变基因表达和染色质的稳健模式。 可访问性，以便能够评估未来的精确治疗。 在申请人看来，这项工作是创新的，因为它侧重于一种新颖的方法， 为重叠的罕见ID疾病组开发共享疗法，并提出了一种独特的染色质 用于未来小分子筛选的可及性测定。这项工作意义重大，因为预计 提供强有力的证据支持扩大其他染色质疾病的方法， 纠正这些染色质破坏的化合物。最终，这些知识有可能提供新的 为智力残疾的创新治疗提供了机会。

PROJECT SUMMARY Over recent several decades, genomic analyses have elucidated etiologies for hundreds of disorders with syndromic and nonsyndromic intellectual disability (ID). However, this knowledge has not readily translated into treatment for patients, partly due to therapy development strategies that focus solely on single diseases. Many patients with ID have mutations in genes that encode chromatin-organizing proteins. Mutations in cohesin, a key chromatin-regulating protein, cause overlapping clinical findings with patients who have mutations in other chromatin regulatory complexes, including BAF/SWI-SNF and p160/NCOA. The long-term goal is to identify treatments to improve neurocognitive outcomes in children with chromatin-regulatory disorders. The overall objective of this R21 is to identify common informative target genes disrupted by chromatin regulatory gene mutations in experimentally approachable and patient-derived cell models. The central hypothesis is that mutations in cohesin, BAF/SWI-SNF and p160/NCOA complexes cause similar clinical features because they disrupt an overlapping set of target genes. The rationale is that identification of common, reproducible chromatin disruptions will enable breakthrough work to rapidly screen drugs in laboratory settings and enable confirmation of precision therapy in patient cells prior to treatment. This central hypothesis will be tested by the following two specific aims: 1) Identify common regions of chromatin disruption in HCT116 cells with heterozygous mutations in cohesin, BAF/SWI-SNF and p160/NCOA complex genes; and 2) Identify common regions of chromatin disruption in patient-derived lymphoblastoid cells. For the first aim, CRISPR-Cas9 will be used to create heterozygous loss-of-function mutations for each of six chromatin-regulatory genes in a monogenic lab cell line. RNA-seq and ATAC-seq will be used to identify resultant patterns of altered gene expression and chromatin accessibility. Under the second aim, the investigators' large collection of heterogenous patient lymphoblastoid cell lines with mutations in three chromatin-regulatory genes will be used to identify robust patterns of altered gene expression and chromatin accessibility to enable assessment of future precision therapy. This work is innovative, in the applicant's opinion, because it focuses on a novel approach to developing shared therapies for overlapping groups of rare ID disorders, and proposes a unique chromatin accessibility assay for future small molecule screening. This work is significant because it is expected to provide strong evidence to support widening the approach to additional chromatin disorders and pursing compounds that correct these chromatin disruptions. Ultimately, such knowledge has the potential to offer new opportunities for innovative treatments of intellectual disability.