Angelman syndrome (AS) is a rare neurological disorder caused by loss of Ube3a protein in neurons. In all individuals, this loss occurs from the paternal allele due to genomic imprinting; at around week 10 of neuronal development, a long noncoding RNA, Ube3a-ATS, is transcribed from the paternal allele. Ube3a-ATS physically overlaps paternal Ube3a, thereby blocking its transcription. Individuals with AS have a complete absence of Ube3a expression in the brain due to a maternal loss of function.
One strategy for molecular rescue in AS is reactivation of paternal Ube3a via repression of Ube3a-ATS. Previous success using this approach has been attained in the lab of our collaborator, Dr. David Segal (UC Davis), using a purified protein murine artificial transcription factor (mATF) to treat a murine model of AS. Unfortunately, the half-life of the protein in the brain was found to be short, requiring injections three times per week. To translate this to a human therapeutic would require large doses of purified protein per week, which is not only costly, but also creates an increased capacity to trigger a patient’s immune response.

The goal of my project is production of a human ATF (hATF) mimicking the design of the mATF and an optimized delivery system which, together, restore paternal Ube3a in two human AS neuronal lines. One line, AGI-O, carries a large-scale deletion in maternal Ube3a. The other, AS_Δ3, has a 3 base pair deletion. I have carried out AGI-O time course experiments which have implicated imprinting as a neuronal maturation phenomenon requiring at least 12 weeks in vitro differentiation. At week 10, neurons display very little expression of Ube3a-ATS and no decrease in Ube3a. However, at weeks 12 and 14, Ube3a-ATS is much more strongly expressed, and a concurrent decrease in Ube3a is seen. This defines a time point at which to test novel therapeutics.

At this time, we have several candidate hATFs undergoing testing on 12-week AGI-O neurons to determine the top reactivator(s) of paternal Ube3a. Once we determine the top candidate(s), we plan to use genetically-modified mesenchymal stem cells (MSCs) as “micropharmacies” to manufacture and secrete our hATF, circumventing the costs of continuous protein purification and evading the immune system via native immunosuppressive properties. We will treat the AS neurons with conditioned media from the MSCs and test for a reactivation of paternal Ube3a via Western blot and RT-PCR for Ube3a and Ube3a-ATS.