Angelman syndrome (AS) is a neurodevelopmental disorder that results from the loss of UBE3A expression from the maternally-inherited allele of human chromosome 15q11-q13. UBE3A encodes an E3 ubiquitin ligase that targets proteins for degradation by the ubiquitin-proteasome pathway. There are three protein isoforms of UBE3A, which differ at their N-termini. We hypothesize that loss of one UBE3A isoform will be primarily responsible for AS phenotypes. We propose to study individual UBE3A protein isoforms using patient- specific human induced pluripotent stem cells (iPSCs) and their neuronal derivatives. We have genetically corrected an AS iPSC line harboring a missense mutation in UBE3A. We will mutate the translational start sites of individual UBE3A protein isoforms to determine their relative abundance and subcellular localization. We will then investigate the cellular phenotypes of neurons with individual isoforms mutated. The studies describe herein to investigate the contribution of individual UBE3A isoforms to AS will contribute important knowledge toward the understanding of neuronal UBE3A function and the development of gene therapies for AS.
These experiments propose to use genome editing to ablate individual UBE3A protein isoforms in human Angelman syndrome (AS) induced pluripotent stem cells to determine the relative expression levels and subcellular localization of each isoform as well as their contribution to the AS cellular phenotype. This study benefits public health because it will identify the cellular compartment in which UBE3A is most important and will identify the most appropriate UBE3A isoform for use in gene therapy approaches.
