Antisense oligonucleotide-based upregulation of neurodevelopmental disorder gene MYT1L

PROJECT SUMMARY Therapeutic restoration of protein function is a key goal for many neurodevelopmental disorders (NDDs) caused by genetic haploinsufficiency. MYT1L syndrome is a recently identified, understudied NDD caused by heterozygous loss of function mutations in the MYT1L gene, characterized by global developmental delay, particularly in motor and language development, intellectual disability, highly penetrant obesity and hypotonia, and a significant comorbidity of autism spectrum disorder (ASD) and/or attention-deficit/hyperactivity disorder (ADHD). Mice and human neurons with a MYT1L stop-gain mutation only show ~30% decreased transcript and protein levels, yet display profound cellular, molecular, and behavioral anomalies, indicating that MYT1L levels need to be tightly controlled for normal function. However, it is unknown how MYT1L levels are regulated. Understanding this regulation is a key step to identifying clinically relevant strategies for upregulating MYT1L as a therapy for MYT1L syndrome. An emerging protein upregulation strategy is to use antisense oligonucleotides (ASOs) to block elements that normally destabilize mRNA. Thus, ASOs could be used to increase protein expression from the mRNA of the remaining healthy allele. MYT1L has a conserved and longer than average 3’ untranslated region (UTR), which contains many regulatory elements responsible for transcript stability and translation efficiency, properties critical for protein synthesis. Using bioinformatic analyses, existing microRNA (miRNA) binding data, and a preliminary screen for active regulatory elements using a massively parallel reporter assay (MPRA), we have identified regions that may reduce stability of MYT1L transcripts, which I have therefore termed MYT1L Negative Regulatory Elements (MNREs), and discovered several candidate ASOs that increase expression. MNREs contain several predicted miRNA response elements (MRE) and Pumilio (PUM) response elements (PRE), which both induce translational repression or transcript degradation. This project seeks to study MYT1L post- transcriptional regulation (PTR) and its potential to be harnessed in translational therapies like ASOs for MYT1L syndrome. Aim 1 will test the importance of specific MRE and PRE sequences in MYT1L mRNA stability and screen for additional MNREs using an MPRA in iPSC-derived neurons to capture PTR in an appropriate cellular context. ASOs will both be used as tools and potentially be deliverables. Aim 2 will test the efficacy of a candidate ASO targeting a conserved MRE in restoring MYT1L protein levels in MYT1L haploinsufficient mice and rescuing associated transcriptomic and behavioral phenotypes. These findings will begin to explore MYT1L PTR and provide clinically relevant insights for rescue of MYT1L haploinsufficiency by direct targeting of MYT1L. This project will also provide the applicant the opportunity to train in RNA therapeutics, computational genomics, animal behavior to prepare for a future career as a physician-scientist studying neurogenetic disorders.