Engineered Chromosomal Copy Number Variations in Human Stem Cells to Model Psychiatric Disorders

工程化人类干细胞染色体拷贝数变异来模拟精神疾病

• 批准号: 272697065
• 负责人: Dr. Daniel Haag
• 金额: --
• 依托单位: Department of Pathology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/272697065?language=en
• 关键词: Engineered; Chromosomal; Copy; Number; Variations

Psychiatric diseases like schizophrenia and autism represent severe neurodevelopmental disorders with an increasing penetrance of currently about 1 percent of the population. The heterogeneous appearance of these diseases is reflected by their complex genetic causes which include rare gene mutations and frequent copy number variations (CNVs) of small chromosomal regions. Although numerous recurrent CNVs have been identified, the investigation of associated mechanisms of disease development is extremely challenging. So far, analyses of targeted genome modifications can only be conducted in mouse models, but inter-species differences in chromosomal architecture and development of certain brain regions make it difficult to translate findings to the human situation. The use of human induced pluripotent stem cells (iPSCs), however, presents a promising alternative for modeling diseases. These stem cells can be genetically modified using versatile gene-targeting techniques and subsequently differentiated into virtual every type of cell. Therefore, disease-associated genetic alterations can be recapitulated in a human context and direct consequences can be analyzed in a relevant tissue. In the outlined project, a CNV of a 500 kilobase region on chromosome 16p11.2 will be generated in human stem cells. Both duplication and deletion of this region are associated with autism-spectrum disorders and the duplication is recurrently found in patients with schizophrenia. Using adeno-associated virus two recombination sequences will be inserted into the genome of the stem cells encasing the CNV region. Upon addition of the enzyme Cre recombinase these recombination sequences can connect with each other and thereby either loop out the region between them or duplicate the same region if different recombination sequences on two sister chromatides interact. By implementing different resistance genes, deletion and duplication events can be specifically selected to generate isogenic cell lines harboring different copy numbers of the candidate region. In order to study the biological consequences on neuronal functions the stem cells will be differentiated into neurons by introducing distinct transcription factors. Induced neurons (iNs) with different CNVs will then be subjected to electrophysiological examination to detect specific effects on synaptic transmission and stimulus propagation. Moreover, analyses of shape and outgrowth behavior of the neurons will be measured and differences in gene expression levels will be analyzed. The aim is to identify molecular changes on a cellular level and to unravel disease-causing processes that are related with the CNV on 16p11.2. The findings from this study should therefore help to develop more specific therapies to target the diverse symptoms of autism and schizophrenia patients more accurately.

精神分裂症和自闭症等精神疾病是严重的神经发育障碍，目前约占人口的1%。这些疾病的异质性表现在其复杂的遗传原因上，包括罕见的基因突变和小染色体区域的频繁拷贝数变异(CNV)。虽然已经发现了许多复发的CNV，但对疾病发展的相关机制的调查是极其具有挑战性的。到目前为止，对定向基因组修改的分析只能在小鼠模型中进行，但染色体结构和某些大脑区域的发育的物种间差异使得很难将发现转化为人类的情况。然而，使用人类诱导的多能干细胞(IPSCs)为疾病建模提供了一个有前途的替代方案。这些干细胞可以使用通用的基因打靶技术进行基因改造，然后分化成几乎所有类型的细胞。因此，与疾病相关的基因改变可以在人类背景下概括，并可以在相关组织中分析直接后果。在概述的项目中，将在人类干细胞中产生染色体16p11.2上500千碱基区域的CNV。该区域的重复和缺失都与自闭症谱系障碍有关，这种重复经常在精神分裂症患者中发现。使用腺相关病毒，两个重组序列将被插入包围CNV区域的干细胞的基因组中。在加入Cre重组酶后，这些重组序列可以相互连接，从而在两个姐妹染色单体上的不同重组序列相互作用的情况下，要么环化它们之间的区域，要么复制相同的区域。通过实施不同的抗性基因，可以特异性地选择缺失和复制事件，以产生具有不同候选区域拷贝数的等基因细胞系。为了研究干细胞对神经元功能的生物学影响，将通过引入不同的转录因子将干细胞分化为神经元。然后对具有不同CNV的诱导神经元(INS)进行电生理检查，以检测对突触传递和刺激传播的特定影响。此外，还将测量神经元的形状和生长行为的分析，并分析基因表达水平的差异。其目的是识别细胞水平上的分子变化，并揭开与16p11.2上CNV相关的致病过程。因此，这项研究的发现应该有助于开发更具体的治疗方法，以更准确地针对自闭症和精神分裂症患者的不同症状。