Splice-modulating antisense oligonucleotides targeting a pathogenic intronic variant in adult polyglucosan body disease correct mis-splicing and restore enzyme activity in patient cells.

Adult polyglucosan body disease (APBD) is a rare, adult-onset neurodegenerative disorder caused by loss-of-function variants in the glycogen branching enzyme (GBE1) gene, essential for glycogen biosynthesis. The second most common pathogenic mutation in APBD (c.2053-3358_2053-3350delinsTGTTTTTTACATGACAGGT) is a deep intronic deletion insertion (indel) variant creating an ectopic splice acceptor site, resulting in a mutant transcript with a pseudoexon encoding an unstable truncated protein. Such mutations can be effectively targeted with splice-modulating antisense oligonucleotides (ASOs) to restore normal splicing. Here, we characterized the indel in-depth using long-read sequencing techniques and discovered several new features of the mutant transcript. The indel sequence varies from the previously identified sequence by a nucleotide, and the usage of the ectopic splice site results in two mutant isoforms, both of which are targets of cellular nonsense-mediated decay. High-throughput screening in patient-derived fibroblasts identified multiple lead candidates that effectively blocked the ectopic splice site and increased the canonical GBE1 transcript and protein. Functional analysis confirmed that treatment with the lead ASOs significantly improved GBE1 enzyme activity in patient cells, validating their therapeutic potential. Taken together, our data demonstrate the successful discovery of ASOs that correct mis-splicing, thus offering a promising treatment for a subset of APBD patients.