Assessment of the functional impact on the pre‐mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology

Abstract

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha‐glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre‐messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.‐32‐13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.

Opportunities and challenges for antisense oligonucleotide therapies

“…Most effort has focused on restoring canonical splicing in patients with Pompe disease that carry the c.-32-13T>G splicing variant. [78][79][80][81] Approximately 80% of Caucasian childhood and adult onset patients carry this variant in intron 1, which decreases the recognition of the splice acceptor site of exon 2 by the splicing machinery ( Figure 2F,G). This results in inclusion of an intronic natural pseudoexon.…”

“…This resulted in restoration of GAA enzyme activity. In another strategy, AONs promoted exon 2 inclusion by targeting exon 2 itself, which most likely involve the suppression of exonic splice silencer elements 80,81 (Figure 2G). This approach may offer an alternative to enzyme replacement therapy for patients with the c.-32-13T>G splicing variant in the future.…”

A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease

“…As a consequence, while both c.546G>T and IVS1 caused exon 2 skipping, AONs targeting distinct cryptic splice sites were required to correct these variants: AONs that blocked the cryptic splice donor and/or acceptor site of a pseudoexon in intron 1 in the case of IVS1 [10,11], and AONs that blocked the cryptic splice donor site at c.546 + 184 in the case of c.546G>T (this study). In addition, AONs targeting exon 2 were found to be effective in restoring aberrant splicing caused by the IVS1 variant but not c.546G>T [12,28]. These findings highlights the importance of functional splicing analysis for diagnostics and the design of treatment options.…”

“…For Pompe disease several AONs have been developed, including AONs targeting rare variants [9] and AONs that prevent aberrant splicing caused by the IVS1 variant [10][11][12]. Furthermore, AONs that prevent aberrant splicing caused by the IVS1 variant can potentially be used to rescue aberrant splicing resulting from other variants present in GAA exon 2 [28]. Here we described the design of a novel AON that targets aberrant splice products generated due to the presence of the c.546G>T variant and enhances GAA enzyme activity in cells carrying this variant.…”

Update of the Pompe variant database for the prediction of clinical phenotypes: Novel disease‐associated variants, common sequence variants, and results from newborn screening

“…All variants had a low CADD score (<10; Table 2) and were classified as "unknown." We note that while these common sequence No effect on splicing of exon 2 in minigene construct (Goina, et al, 2019) Taiwan; Chien et al (2011) c.546+5G>T* p.? 10% residual activity of GAA in expression study (Kroos, et al, 2012a) Taiwan; Chien et al (2011) c.1805C>T (Kroos et al, 2006;Labrousse et al, 2010).…”