Oligonucleotides targeting the 3′ splice site downstream of a microexon as an innovative therapy for autism

Microexons are enriched in genes linked to autism spectrum disorders (ASDs). We have previously demonstrated decreased inclusion of a neuronal specific 24 bp microexon of the translational regulator <i>CPEB4</i> in brains of idiopathic ASD cases and that this leads to CPEB4 aggregation and subsequent under-expression of multiple high confidence ASD-risk genes. Furthermore, decreased inclusion of the <i>CPEB4</i> microexon is also a novel etiological mechanism in schizophrenia (SCZ). In this work, we designed a series of splice switching antisense oligonucleotides (SSOs) targeting the <i>CPEB4</i> microexon genomic region. SSOs targeting intronic regions near the microexon resulted in a decrease in microexon inclusion in neuroblastoma cells, by blocking hnRNPC binding, mimicking the isoform imbalance observed in ASD. Interestingly, we identified SSOs targeting downstream 3' splice site of exon 5 that favoured microexon inclusion in a dose-dependent manner and resulted in increased protein levels of <i>AUTS2</i> and <i>DIRK1A</i>, two high-confidence ASD risk genes, targets of CPEB4, with reduced protein levels in ASD. These results deepen our understanding of the complex splicing regulation of microexons and open new applications of SSOs to treat diseases, such as ASD and SCZ, characterized by altered microexon inclusion.