Developing Therapeutic Splice-Correcting Antisense Oligomers for Adult-Onset Pompe Disease with c.-32-13T>G Mutation.

The mutation c.-32-13T>G in the GAA gene impacts normal exon 2 splicing and is found in two-thirds of late-onset Pompe disease cases. We have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients carrying this common mutation. We performed in silico analysis of the GAA gene transcript for potential splicing silencers and designed oligomers targeting motifs predicted to enhance exon 2 retention in the mature mRNA. Two patient-derived fibroblasts were obtained from Coriell Institute for Medical Research, and seven fibroblast strains from unrelated patients were supplied by Westmead Hospital in Sydney, Australia. Both fibroblasts and forced-myogenic cells were treated with optimized phosphorodiamidate morpholino oligomers supplied by Sarepta Therapeutics. Total RNA and protein were extracted from the cells after incubation with phosphorodiamidate morpholino oligomers, and RT-PCR and RT-qPCR were performed to confirm exon 2 inclusion is enhanced. Acid α-glucosidase activity and expression levels were also assessed to confirm therapeutic potential.