Modulating splicing in 5ʹ untranslated regions to treat rare haploinsufficient disease

Abstract Background Rare genetic disorders collectively impact over 300 million people worldwide, yet around 95% have no specific treatments. For the many rare disorders caused by haploinsufficiency, effective therapies need to upregulate protein expression. However, therapeutic upregulation is often not straightforward. Increasing protein translation from the wildtype allele through inhibiting repressive upstream open reading frames (uORFs) has been proposed as a therapeutic approach for a few specific genes. The widespread success of steric-block antisense oligonucleotides (ASOs) for this purpose is, however, debated. Methods Here, we explore an alternative approach, using splice-switching to exclude uORF-containing exons from the mRNA. We defined 2,210 potentially ‘skippable’ 5ʹUTR exons using MANE transcript definitions. uORF start codons in these exons were identified using ribosome profiling data from 13 human tissues and cell lines, including brain, heart and skeletal muscle. The effect of skipping six prioritised candidate target exons on downstream translation was assessed using dual luciferase assays. Results We identified 1,056 skippable 5ʹUTR exons containing uORF start codons, 79 of which were in haploinsufficient monogenic disease genes. We prioritised six candidate 5ʹUTR exons in neurodevelopmental disorder genes and demonstrate that removing the target exon significantly increased protein translation (between 1.4 to 5.5-fold) for four of the six prioritised genes ( CTCF , GRIN2B , KRIT1 , and TSC1 ). Further, we show that this effect is likely primarily driven by the uORF rather than other regulatory factors within the target exons, as removing the uORF start sites alone increased protein translation to comparative levels (between 1.4 to 7.9-fold). For a single gene target ( TSC1 ), we show that splice-switching ASOs can elicit target exon skipping and up-regulate endogenous protein levels. Further, we show that TSC1 uORFs act additively to reduce protein translation. Conclusions We show that skipping 5ʹUTR exons can boost protein translation. This supports the broad application of 5ʹUTR exon skipping as a therapeutic mechanism to upregulate protein production from clinically relevant haploinsufficient genes.