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

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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.