Angelman syndrome patient neuron screen identifies a potent and selective clinical ASO targeting UBE3A-ATS with long lasting effect in cynomolgus monkey

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A with no available treatment. Restoring UBE3A levels via downregulation of the paternally cis-acting long non-coding antisense transcript (UBE3A-ATS) is a potential disease modifying. Developing molecules targeting human UBE3A-ATS is challenging because it is expressed only in neurons and lacks animal species sequence conservation. To overcome this, we performed a library screen of locked-nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) on AS patient-derived neurons, identifying initial sequences. Further optimization led to the identification of the ASO, RO7248824, which selectively and potently reduces UBE3A-ATS, while concomitantly upregulating the UBE3A mRNA and protein. These properties held true in both human AS patient- and neurotypical-, as well as cynomolgus monkey-derived neurons. In vivo use of tool molecules in wild-type (WT) and AS Ube3am-/p+ mice, revealed a steep relationship between UBE3A-ATS knock-down and UBE3A mRNA/protein upregulation, whereby an almost 90% downregulation was needed to achieve a 50% upregulation, respectively. This relationship was confirmed in cynomolgus monkeys. Whereby, repeated lumbar intrathecal administrations of RO7248824 was well tolerated without adverse in-life effects or tissue pathology and produced a robust, long lasting (up to 3 months) paternal reactivation of UBE3A mRNA/protein across key monkey brain regions. Our results demonstrate that AS human pluripotent stem cell neurons serve as an excellent translational tool and furthermore LNA-modified ASOs exhibit excellent drug-like properties. Sustained efficacy translated to infrequent, intrathecal dosing and serves as the basis for the ongoing clinical development of RO7248824 for AS. ![Graphical abstract.][1] Graphical abstract. From AS patient blood to a neuronal screen, identifies clinical ASO with excellent in vivo properties. ( 1 ) Patients were recruited. ( 2 ) Whereby blood was reprogrammed into hIPSC and subsequently differentiated into neurons. ( 3 ) ASOs were designed and screened on human neurons to downregulate the UBE3A-ATS likely via directed RNase H Cleavage of Nascent Transcripts. ( 4a ) RO7248824 was identified that potently and selective reduces UBE3A-ATS, concomitantly with upregulating the UBE3A sense transcript and protein which was used for in vitro pk/pd. ( 4b ) In parallel tool murine ASO were used demonstrate in vivo POC.(5) Pivotal nonhuman primate studies to monitor safety and predict the human dose. (6) RO7248824 is in AS clinical trial . One Sentence Summary From angelman syndrome human neuron screen to cynomolgus monkey proof of concept identifies the clinical molecule RO7248824 ### Competing Interest Statement S.R., C.B., E.K., S.B., M.B., M.P., D.T., L.P., M.M., K.D., M.T., L.J., J.H., N.J.P., V.C., T.K., A.B., L.M., A.B-B, R.J., M.C.H., were or are employed by F. Hoffmann-La Roche. Parts of the work in this study have been filed in the patent application WO2017/081223A1. [1]: pending:yes