Relevance of functional studies for assessing an antisense oligonucleotide-mediated exon skipping therapeutic strategy for mucolipidosis type II

Abstract Background Mucolipidosis type II (ML II) is a lysosomal storage disorder caused by deficiency of N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT), which impairs the trafficking of lysosomal hydrolases. Of all ML II pathogenic variants, c.3503_3504del in GNPTAB exon 19 is the most prevalent, therefore constituting a compelling molecular target for the development of personalized therapeutic strategies. Here, we explored the feasibility of an innovative RNA based-therapeutic strategy using antisense oligonucleotides (ASOs) designed to induce exon 19 skipping in GNPTAB pre-mRNA. This approach was previously successfully tested at the mRNA level in fibroblasts from ML II patients, where it generated an in-frame mRNA. In the present study, our aim was to evaluate whether GNPTAB exon 19 skipping could increase GlcNAc-PT levels and consequently improve the cellular phenotype of ML II patients carrying this pathogenic variant. To address this, we designed a functional approach based on the overexpression of a GNPTAB construct carrying the exon 19 deletion enabling the indirect evaluation of the resulting protein´s functionality. Results Our first results demonstrated that in ML II fibroblasts, ASO treatment led to a modest increase in lysosomal hydrolase activity at 24 h and 48 h. Moreover, LAMP-1 expression remained elevated and comparable to untreated ML II cells, indicating that GlcNAc-PT activity was not restored. To further investigate the functional relevance of exon 19 skipping, overexpression studies were performed in HEK293T cells. Three constructs (pGNPTAB WT, pGNPTAB del_exon19 and pGNPTAB c.3503_3504del) were expressed. Both pGNPTAB WT and pGNPTAB del_exon19 constructs produced the α/β-precursor. However, only the WT construct generated the mature β-subunit, whereas the pGNPTAB c.3503_3504del construct showed no detectable expression. These findings indicate that exon 19 is essential for proper GlcNAc-PT processing and enzymatic activity. Conclusions Although ASO treatment corrected splicing at the mRNA level, it did not restore GlcNAc-PT activity in ML II patient cells. Nonetheless, our findings clarify the functional importance of exon 19 and demonstrate that overexpression of an exon-skipped construct provides a simple and effective strategy to indirectly assess protein functionality, supporting the prioritization of this kind of approach to test an exon-skipping ASO-based approach before advancing to studies in patient-derived cells.