Luminescence-based biosensor to non-invasively measure UBE3A activity in the brain

SUMMARY Genetic mutations that alter UBE3A ubiquitin ligase activity are linked to several neurodevelopmental disorders. Angelman syndrome (AS) is a severe syndromic neurodevelopmental disorder that is caused by deletion or loss- of-function (LOF) mutation of the maternally inherited copy of UBE3A. In contrast, duplication of maternal or paternal UBE3A is associated with Dup15q syndrome, one of the most common forms of autism spectrum disorder. And gain-of-function (GOF) missense mutations in UBE3A cause neurodevelopmental phenotypes that are distinct from AS in humans and in mouse models. Therapeutics are being developed to increase or decrease UBE3A as treatments for AS and Dup15q, respectively. While reporter assays exist to evaluate UBE3A activity, these assays cannot be used to measure endogenous UBE3A activity in cells or in the brain non-invasively over time. This substantially limits the speed at which therapeutics that normalize UBE3A levels in vivo can be tested and advanced for these debilitating disorders. In preliminary studies, we developed a highly sensitive luciferase- based biosensor that can quantify endogenous UBE3A activity in cells and primary neurons. This biosensor can also quantify the spectrum of UBE3A activity associated with UBE3A LOF and GOF missense mutations. Here, we made additional modifications to the biosensor to improve its sensitivity to changes in UBE3A activity. We hypothesize that this biosensor with improved sensitivity can be used to quantify aberrant UBE3A activity in cells and in the brain of live animals that model various forms of autism. We propose to evaluate the efficacy of this UBE3A biosensor in primary cells (neurons, fibroblasts, blood cells) and in the brain of mouse lines that model AS (maternal Ube3a deficiency), Ube3a gene duplication, and an autism-linked Ube3a GOF mutation. We will also generate and validate a knock-in mouse harboring this UBE3A biosensor expressed from a neuron-specific promoter. This UBE3A biosensor knock-in mouse line could be used in future studies to non- invasively monitor the efficacy and durability of therapeutics that increase or decrease UBE3A activity, as respective treatments for AS and various forms of autism.