Generation of Isogenic Human Pluripotent Stem Cell-Derived Neurons to Establish a Molecular Angelman Syndrome Phenotype and to Study the UBE3A Protein Isoforms

Angelman Syndrome (AS) is a neurodevelopment disorder for which there is currently no cure that is characterized by severe seizures, intellectual disability, absent speech, ataxia, and happy affect. Loss of expression from the maternally inherited copy of UBE3A, a gene regulated by genomic imprinting, causes AS. Currently there are multiple promising therapeutic approaches being explored and developed for AS, some of which involve targeting or expression of the human genetic sequence. Subsequently, it is necessary to establish robust cellular models for AS that can be used to test these, as well as future, potential AS therapies. Toward this aim, here we have used the CRISPR/Cas9 genome editing system to generate several isogenic human pluripotent stem cell lines two achieve two primary goals. First, we aimed to establish a robust quantitative molecular phenotype for cultured human AS neurons using the transcriptome. We identified and validated a list of genes that are consistent differentially expressed in AS neurons when compared to isogenic controls that can be assayed following drug treatments. Second, we aimed to study the abundance and localization of the three human UBE3A protein isoforms. We found that isoform 1 is the predominant protein isoform, and that UBE3A, regardless of isoform, appears to localize mostly to the cytoplasm, with low levels of expression in the nucleus and other organelles. The work in this thesis demonstrates that differentially expressed genes can be used as a phenotype for AS neurons to measure the effects of potential therapies, and provides important and previously unknown information as to the abundance and localization of the human UBE3A protein isoforms in human neurons