Abstracts

Rationale: Chromosome 15q duplication syndrome (Dup15q) is a neurodevelopmental disorder in which patients present with language impairments, hypotonia, intellectual disability, and seizures – which occur in most patients, are often intractable, and result in an increased risk of sudden unexpected death in epilepsy. Despite the substantial unmet medical need, there is currently no disease-specific treatment for Dup15q. Although >30 genes are located within the duplicated region, UBE3A, a gene encoding an E3 ubiquitin ligase, is a critical pathogenic factor.
_x000D_ Antisense oligonucleotides (ASOs) have emerged as a powerful therapeutic modality. Intrathecal administration of ASOs permits direct targeting of the central nervous system (CNS), and the approval of Nusinersen for spinal muscular atrophy has paved the way for the intrathecal ASO modality as a therapeutic approach for genetic disorders of the CNS. Gapmers represent a class of ASOs designed to engage endogenous RNase H, leading to specific degradation of the target transcript. Here we focus on developing ASO gapmers to target knockdown of excess UBE3A in Dup15q as a way of achieving therapeutic benefit. The overall objective of this program is to bring a much-needed, genetically-targeted precision therapeutic to Dup15q patients, and their families and caregivers.

Methods: Here, we identified candidate ASOs for Dup15q using our BRITE^TM system, which integrates patient-derived neuronal models, multi-omics characterization of neuronal cell types, and machine-learning based analytics to uncover disease relevant cellular phenotypes and to quantitatively assess on and off-target ASO activity. Using our in-house ASO design methodology, we generated gapmer ASOs targeting UBE3A, without off-target interference of critical genetic and cellular pathways. ASOs were then characterized using qPCR, immunoblotting, and RNA-sequencing; functional characterization was performed with our proprietary all-optical electrophysiology.

Results: UBE3A-targeting ASOs passed stringent filters to avoid binding to off-target transcripts, undesired thermodynamic properties, and sequence motif liabilities. We identified ASOs achieving >80% UBE3A transcript and protein knockdown that are de-risked to avoid perturbation of neuronal physiology. Based on this characterization, we selected optimal UBE3A ASO leads for assessment in vivo, demonstrating initial rodent tolerability and knockdown of Ube3a in different brain regions using intrathecal delivery.

Conclusions: ASOs were assessed across diverse cell types, including control and Dup15q patient fibroblasts, iPS cell-derived cortical excitatory neurons, rodent primary cortical neurons, and non-human primate primary fibroblasts. Our results indicate we have efficacious, de-risked, and well-tolerated ASOs that engage Ube3a in vivo that will move forward for therapeutic development including demonstration of functional rescue of human phenotypes identified from Dup15q patient-derived models and further in vivo characterization.

Funding: None