OLIGONUCLEOTIDES TARGETING THE SPLICE ACCEPTOR SITE DOWNSTREAM OF A MICROEXON AS AN INNOVATIVE THERAPY FOR AUTISM

Abstract

BackgroundMicroexons are highly conserved and mostly neuronal-specific 3-27 nucleotide exons, enriched in genes linked to autism spectrum disorders (ASD). We have previously shown decreased inclusion of a neuronal specific 24 bp microexon (exon 4) of the translational regulator CPEB4 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, enhanced skipping of the CPEB4 microexon is also a novel etiological mechanism in schizophrenia (SCZ). MethodsIn this work we designed and tested in neuroblastoma cells a series of splice switching antisense oligonucleotides (SSO) targeting the CPEB4 genomic region surrounding the microexon. ResultsSSOs targeting candidate intronic regions near the microexon resulted in a decrease in microexon inclusion by blocking hnRNPC/PTPB1 binding, thus mimicking the isoform imbalance observed in ASD. However, based on the kinetic coupling model correlating transcriptional elongation with splicing regulation, 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 FOXP1 and AUTS2, two high-confidence ASD risk genes that are known targets of CPEB4 and whose protein levels are reduced in ASD. ConclusionsThese 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 that are characterized by altered microexon inclusion.