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

精神分裂症和自闭症等精神疾病代表了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为其复杂的遗传原因，包括罕见的基因突变和频繁的小染色体区域拷贝数变异（CNVs）。尽管已经发现了许多复发性CNVs，但对疾病发展的相关机制的研究极具挑战性。到目前为止，对靶向基因组修饰的分析只能在小鼠模型中进行，但物种间染色体结构和某些大脑区域发育的差异使得很难将研究结果转化为人类的情况。然而，人类诱导多能干细胞（iPSCs）的使用为疾病建模提供了一种有希望的替代方法。这些干细胞可以使用多种基因靶向技术进行基因修饰，随后分化成几乎所有类型的细胞。因此，疾病相关的遗传改变可以在人类环境中重现，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，并且在精神分裂症患者中经常发现重复。利用腺相关病毒将两个重组序列插入包裹CNV区域的干细胞基因组中。在加入酶Cre重组酶后，这些重组序列可以相互连接，从而环出它们之间的区域，或者如果两个姐妹染色单体上不同的重组序列相互作用，则复制相同的区域。通过植入不同的抗性基因，可以特异性地选择缺失和复制事件来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，需要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CNVs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，还将分析神经元的形状和生长行为，并分析基因表达水平的差异。目的是确定细胞水平上的分子变化，并揭示与16p11.2上的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各种症状。

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.