Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease

Steve D Wilton,Russell Johnsen,Frederick J Schnell,May Thandar Aung-Htut,Michel Tchan,Kristin A Ham,Sue Fletcher

doi:10.1038/s41598-020-63461-2

Abstract

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-α-glucosidase activity in patients, and a progressive decline in mobility and respiratory function. Enzyme replacement therapy is one therapeutic option, but since not all patients respond to this treatment, alternative interventions should be considered. One GAA mutation, c.-32-13T > G, impacts upon normal exon 2 splicing and is found in two-thirds of late-onset cases. We and others have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients with one c.-32-13T > G allele. We designed 20 oligomers and treated fibroblasts derived from five patients to identify an oligomer sequence that maximally increased enzyme activity in all fibroblasts. The most effective splice correcting oligomer was chosen to treat forced-myogenic cells, derived from fibroblasts from nine patients carrying the c.-32-13T > G mutation. After transfection, we show increased levels of the full-length GAA transcript, acid-α-glucosidase protein, and enzyme activity in all patients’ myogenic cells, regardless of the nature of the mutation in the other allele. This data encourages the initiation of clinical trials to assess the therapeutic efficacy of this oligomer for those patients carrying the c.-32-13T > G mutation.

Highlights

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-αglucosidase activity in patients, and a progressive decline in mobility and respiratory function
Since Pompe disease arises from an insufficiency of the GAA enzyme, enzyme replacement therapy (ERT) is one therapeutic option
Lower levels of the full-length GAA transcript were consistently evident in these patients after amplification across exons 1 to 5 using RT-PCR and qPCR analysis (Fig. 1a)

Summary

Introduction

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-αglucosidase activity in patients, and a progressive decline in mobility and respiratory function. We show increased levels of the full-length GAA transcript, acid-α-glucosidase protein, and enzyme activity in all patients’ myogenic cells, regardless of the nature of the mutation in the other allele This data encourages the initiation of clinical trials to assess the therapeutic efficacy of this oligomer for those patients carrying the c.-32-13T > G mutation. The late-onset form of Pompe disease, known as glycogen storage disease type II (GSD II), presents as a suitable candidate for AO therapy, since approximately two-thirds of the adult Pompe patients harbour a common disease-causing mutation: c.-32-13T > G7 The incidence of this variant is higher in Caucasians and identified in ninety percent of the adult-onset Pompe patients[8]. The first human, open-label, phase 1/2 trial for combination therapy of a modified GAA, in conjunction with a small molecule pharmacological chaperone, has been initiated (NCT02675465, https://clinicaltrials.gov)

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Results

Conclusion